United States Office of Water EPA 440/5-80-055
Environmental Protection Regulations and Standards October 1980
Agency Criteria and Standards Division
Washington DC 20460
vvEPA Ambient
Water Quality
Criteria for
Hexachlorocyclopentadiene
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AMBIENT WATER QUALITY CRITERIA FOR
HEXACHLOROCYCLOPENTADIENE
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
.v-rol iVj::;tr,ion Agency
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
David J. Hansen, ERL-Gulf Breeze
U.S. Environmentla Protection Agency
Mammalian Toxicology and Human Health Effects:
Mary Ann Bell Zanetos (author)
Battelle Columbus Laboratory
Terence M. Grady (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Jerry F. Stara, (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Patrick Durkin
Syracuse Research Corporation
Renate Kimbrough
Center for Disease Control
Robert McGaughy*
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Myron Men1man
Mobil Oil Corporation
Joseph Borzelleca
Medical College of Virginia
Robert Bruce, ECAO-RTP
U.S. Environmental Protection Agency
Fred Coulston
Albany Medical College
Alfred Garvin
University of Cincinnati
James Lucas, HERL
U.S. Environmental Protection Agency
H.M. Mehendale
University of Mississippi Medical
Center
Herb Pahren, HERL
U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, P. Gray, R. Rubinstein.
*CAG Participating Members: Elizabeth L. Anderson, Larry Anderson, Dolph Arnicar,
Steven Bayard, David L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman,
Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosenblatt,
Dharm V. Singh, and Todd W. Thorslund.
IV
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TABLE OF CONTENTS
Page
Criteria Summary
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Chronic Toxicity B-2
Plant Effects B-2
Residues B-3
Miscellaneous B-3
Summary B-3
Criteria B-4
References B-10
Mammalian Toxicology and Human Health Effects C-l
Introduction C-l
Exposure C-l
Ingestion from Water C-l
Ingestion from Food C-l
Inhalation C-3
Dermal C-3
Pharmacokinetics C-4
Effects C-8
Acute, Subacute, and Chronic Toxicity C-8
Synergism and/or Antagonism C-25
Teratogenicity C-25
Mutagenicity C-26
Carcinogenicity C-32
Epidemiologic Studies C-40
Criterion Formulation C-58
Existing Guidelines and Standards C-58
Special Groups at Risk C-59
Basis, and Derivation of Criterion C-60
References C-66
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CRITERIA DOCUMENT
HEXACHLOROCYCLOPENTADIENE
CRITERIA
Aquatic Life
The available data for hexachlorocyclopentadiene indicate that acute and
chronic toxicity to freshwater aquatic life occur at concentrations as low
as 7.0 and 5.2 yg/1, respectively, and would occur at lower concentrations
among species that are more sensitive than those tested.
The available data for hexachlorocyclopentadiene indicate that acute
toxicity to saltwater aquatic life occurs at concentrations as low as 7.0
jig/1 and would occur at lower concentrations among species that are more
sensitive than those tested. No data are available concerning the chronic
toxicity of hexachlorocyclopentadiene to sensitive saltwater aquatic life.
Human Health
For comparison purposes, two approaches were used to derive criterion
levels for hexachlorocyclopentadiene. Based on available toxicity data, for
the protection of public health, the derived level is 206 ug/1. Using
available organoleptic data, for controlling undersirable taste and odor
quality of ambient water, the estimated level is 1 wg/1. It should be rec-
ognized that organoleptic data as a basis for establishing a water quality
criteria have limitations and have no demonstrated relationship to potential
adverse human health effects.
VI
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INTRODUCTION
Hexachlorocyclopentadiene, (hex; C-56; 1,2,3,4,5,5-hexachlorocyclopenta-
diene) is a pale-to-greenish-yellow liquid with the molecular formula,
CgClg. Other physical properties include a molecular weight of 272.77;
a solubility in water of 0.805 mg/1; a vapor pressure of 1 mm Hg at 78-79'C
and a density of 1.7119 (20°/4*C) (Lu, et al. 1975; Ungnade and McBee, 1958).
Hex was the key intermediate in the manufacture of the organochlorine
pesticides endosulfan and PentaiS) and formerly in the manufacture of sev-
eral commercially important organochlorine pesticides whose usage is now
banned or restricted (Kirk and Othmer, 1964). Although it has also been
suggested for use as an intermediate in the manufacture of dyes, Pharmaceu-
ticals, resins, and germicides, these latter uses account for only a very
small percentage of hex production. Historically, hex has been produced in
the United States by two companies, Hooker Chemical and Plastics Corporation
(Montague, Michigan) and Velsicol Chemical Corporation (Memphis, Tennes-
see). In 1977, Hooker discontinued hex manufacture at the Montague plant,
making Velsicol's Memphis plant the only current U.S. producer. Hex is pro-
duced at several facilities outside the U.S. Hex was formerly used in the
manfacture of aldrin, endrin, and dieldrin at the Shell Chemical Company
plant in Denver, Colorado (Zavon, 1978).
Hex has been used as a chemical intermediate in the production of numer-
ous chlorinated -pesticides, several of which have enjoyed very large usage.
The list includes chlordane, aldrin, dieldrin, heptachlor, isodrin, endrin,
mirex, Kepone, endosulfan (ThiodarnO, and PentafcS). With the exception
of endosulfan and PentaoH both of which are in current use, the usage of
hex-based pesticides has been banned, suspended, or severely restricted by
A-l
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governmental action. Although current production estimates are uncertain
and highly variable, one estimate has placed annual production as high 50
million pounds (25,000 tons) ,per year (Bell, et al. 1978). Recent bans or
restrictions on many of the chlorinated pesticides have led to a decline in
the use of hex as a chemical intermediate in the manufacture of these prod-
ucts; simultaneously, the use of hex in the manufacture of flame retardants
has increased. Currently, a major use of hex is in the manufacture of flame
retardant compounds such as chlorendic acid and chlorendic anhydride which
are produced by reacting equimolar quantities of hex and maleic anhydride.
These and other hex-derived chlorinated organic compounds confer flame re-
tardant properties to plastics, including polypropylene, polyethylene, ny-
lon, rigid polyurethane foams, unsaturated polyesters, and other polymers
including eposy resins (Sanders, 1978).
Although hex is a commercially important chemical intermediate with high
annual production, it has essentially no end uses of its own. Consequently,
hex concentrations in the environment should be negligible and limited data
suggest that this indeed is the case. Small amounts of hex are occasionally
present as impurities in pesticides made from it and some has undoubtedly
entered the environment in this way. The most likely route of entry into
the environment arises from the manufacture of hex or hex-containing prod-
ucts. Discharge of these industrial wastes appears to constitute the only
documented sources of measurable hex in environmental samples.
Due to its infrequency in the environment and its low profile as a chem-
ical intermediate, there have been few studies of the behavior of hex in the
environment or in biological systems. By the same token, until recently,
hex was not recognized as a major environmental problem nor a potential
threat to humans (except for those occupationally exposed). A recent inci-
A-2
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dent in which eighty-six workers at a sewage treatment plant in Louisville,
Kentucky, experienced a variety of toxic symptoms following the improper
disposal of hex manufacturing,wastes has created a great demand for informa-
tion concerning the effects of hex exposure on humans.
Several literature reviews on the health and environmental effects of
hex are available. These include reviews of Equitable Environmental Health,
Inc. (1976); U.S. Environmental Protection Agency (1977); National Academy
of Sciences (1977); and Bell, et al. (1978). Although each of these reports
is different in emphasis, they each note the unfortunate absence of epidemi-
ologic studies of hex-exposed workers and the lack of suitable chronic expo-
sure studies of animals (especially with respect to carcinogenicity). Until
these types of information are available, proposed environmental criteria
will necessarily be based on extrapolation of animal data to humans, a prac-
tice which is invariably speculative and prone to error. Perhaps more im-
portantly, in the absence of suitable chronic exposure studies, recommenda-
tions must be based on avoidance of relatively overt manifestations of tox-
icity (e.g., abnormalities in physiologic tests, increased incidence of neo-
plasms, etc.) which may manifest themselves only after years of exposure.
Since effects of the latter type tend to be elicited at doses lower than
those causing acute toxicity, criteria based on acute responses may fail to
provide adequate protection. Consequently, the criterion levels suggested
in this document are presented with the understanding that they are based on
decidedly inadequate chronic effects data and should be reassessed upon com-
pletion of appropriate chronic studies.
Several transport and fate processes appear to operate at significant
rates to remove hexachlorocyclop,entadiene from aquatic systems. The rela-
tive importance of these processes is thought to depend strongly on the
A-3
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characteristics of the individual water body, so that there is not clear
indication that one process is predominant on an overall basis. The most
important fate processes appear to be hydrolysis and near-surface photoly-
sis, while transport occurs via the water column (as dissolved species), by
volatilization to the atmosphere, and through absorption onto particulates
(perhaps to a lesser extent). The fate of hex in the troposphere is not
known (U.S. EPA, 1979).
Six active chlorines and two double bonds make hex a highly reactive
compound which readily undergoes substitution and additional reactions. Its
versatility is based upon its reactivity as a diene with a variety of ole-
fins and polynuclear aromatic hydrocarbons in the Diels-Alder reaction.
Rieck's report (1977a) provides evidence of the volatilization of hex
from soil. Vapors of *4C-hex were evolved from treated soil to the extent
of 11, 13, 15, 16, 17, and 19 percent (cumulative) of the applied amounts,
1, 2, 3, 5, 7, and 14 days respectively after treatment. One could, there-
fore, deduce that there is volatility from treated soil and that the rate
decreases with time.
Another distinguishing feature of hex is that it appears to be strongly
adsorbed to soil or soil components. Two studies of hex-treated soil
(Rieck, 1977a,b) have demonstrated poor extractability from soil, which
provides indirect evidence of strong adsorption. In one study (Rieck,
1977b), soil which had been extracted was then combusted to 1 C02. Any
residual but unextracted * C was then measured directly. Unextracted
*4C was found in these samples and thus was accounted for as a "bound"
residue. Had it not been accounted for, it would have probably been assumed
to have volatilized.
A-4
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Hex, unlike some of the pesticides derived from it, degrades rapidly by
photolysis, giving water soluble degradation products. Tests on its stabil-
ity towards hydrolysis-^t ambient temperature indicated a half-life of about
11 days at pH 3-6, which was reduced to 6 days at pH 9. In another experi-
ment (Bevenue and Yeo, 1969), the vaporization and absorption properties of
hex in organic solvent (iso-octane) and in aqueous media were examined pre-
paratory to studying the adsorptive effects of the chemicals on stored
foods. Gas chromatographic data from the solutions of distilled water con-
taining the adsorbed vapor of hexachlorocyclopentadiene revealed that the
chemical had completely disappeared after three days exposure, indicating
dissipation or decomposition.
Data from the iso-octane solutions revealed no degradation after 24
hours, but a multi-peak spectrum indicating the presence of degradation
products was obtained after 7 to 21 days' exposure. This spectrum suggested
to the investigators that the compound may be susceptible to atmospheric ox-
idation and/or photodecomposition (National Cancer Institute, 1977).
In using hex as an intermediate in the manufacture of various chlorinat-
ed pesticides (chlordane, dieldrin, heptachlor, etc.), it appears that al-
though yields in all reactions are good, they are not quantitative. Thus,
there is reason to suspect that in some cases free hex may have been present
in the marketed pesticide products. An early study by Ingle (1953) provided
evidence that the reported vapor toxicity of chlordane to mice was not at-
tributable to chlordane, but to some unreacted intermediates, chief of which
was hexachlorocyclopentadiene. It is suspected that small quantities of un-
reacted hex may be present in other related pesticides as well.
Because of the widespread use of hex as an intermediate, and the belief
that hex may have at one time comprised as much as one percent of commercial
A-5
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chlordane (Ingle, 1953), laboratory studies have been undertaken to deter-
mine its fate under various environmental conditions (Metcalf, et al. 1971;
Lu, et al. 1975). limited studies indicate that the chemical would not be
expected to persist in the environment. For example, the bioaccumulation
and degradation of C-labeled hex was investigated in a laboratory model
ecosystem, simulating the application of chemicals to plants and subsequent
contamination of the aquatic environment. A 5.0 mg quantity of the labeled
hexach1orocyc1epentadiene was topically applied to plants in the terrestrial
portion of the model and the products were allowed to pass through the en-
tire system over a 33-day period of 80"F and with a 12-hour light cycle.
The concentration of hex reached a maximum level of only 0.031 mg/1 in the
water phase of the model after 14 days, decreasing to 0.016 mg/1 by 33
days. There was evidence of bioaccumulation of hex as indicated by its re-
covery as 33 percent of the extractable C in algae, 50 percent in snail,
46 percent in mosquito, and 41 percent in fish; but the concentration of
total C in these various organisms was relatively low (compared to the
other chemicals tested), indicating substantial volatility. None of the
trace degradation products was identified although the extent of total deg-
radation was estimated to be: water, 77 percent; algae, 4 percent; snail, 10
percent; mosquito, 2 percent; and fish, 37 percent. The interpretation of
this data, especially with respect to biomagnification has been the subject
of controversy (Whitacre, 1978).
Hex enters the environment primarily through discharges and emissions
from pesticide production facilities; smaller quantities enter the environ-
ment through the use of pesticides and compounds in which hex is present as
an impurity, e.g., chlordane (Harris, 1972). Once in the environment it may
be transported by wind, surface and underground water, streams, and biota.
A-6
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In December, 1975, hex was qualitatively identified as a contaminant in
the discharge of a pesticide production plant in Memphis. Later, (May,
1977), the compound was identified in the air at the Hooker plant in Monta-
gue, Michigan (56 ppb), in its aaueous discharge (0.170 mg/1), and in fish
tissue from the receiving stream (14-18 ppb) (Spehar, et al. 1977). Hex has
also been reported to have been present in soil and bay sediments in the
vicintiy of a Virginia pesticide plant long after production was discontin-
ued (Swanson, 1976).
Data on environmental concentrations of hex are minimal except for in-
dustrial discharges. Velsicol Chemical Corporations's Memphis plant has
been issued a National Pollutant Discharge System (NPDS) permit. Monitoring
activities in connection with the discharge permit indicate that hexachloro-
cyclopentadiene, hexachloronorbornene, and hexachlorobornadiene are being
discharged into the City of Memphis wastewater collection system (Bennette,
1977; Marks, 1977). A sampling from the month of January, 1977 (31 consecu-
tive days), revealed hex concentrations in wastewater ranging from 0.156 to
8.240 mg/1. The U.S. Environmental Protection Agency's Water Surveillance
Branch sampled Velsicol's discharge February 2-3, 1977. Hex was detected at
18 mg/1. Based on the average monthly discharge by the Velsicol Chemical
Corporation during February, 1977 (3.16 million gallons per day), 474 pounds
of hex were believed to have been discharged through Velsicol's discharge
outfall into the City of Memphis Wastewater Collection System and then into
the Mississippi River during the period February 2-3, 1977. Calculated on
the basis of the flow rate above, this discharge caused a concentration of
hex in the Mississippi River of 0.0006 mg/1 (Carter, 1977).
In a recent, well-publicized- incident, an estimated 6 tons equivalent of
hexachlorocyclopentadiene (hex) and octachlorocyclopentene (octa) dispersed
A-7
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in No. 4 fuel oil were dumped into the Louisville, Kentucky, municipal sewer
system's Western Outfall sewer. The contaminated sludge entered the Morris
Forman Wastewater Treatment P/lant on March 26, 1977, causing illness among
sewage treatment plant workers. Toxic effects associated with this episode
forced closure of the plant with subsequent diversion of 105 million gallons
per day of raw sewage into the Ohio River. There was no evidence of envi-
ronmental release (outside the immediate environs of the sewage treatment
plant and contaminated sewer lines). It was, however, necessary to decon-
taminate the sewer system and the treatment plant.
A-8
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REFERENCES
Bell, M.A., et al. 1978. Review of the environmental effects of pollut-
ants. XI. Hexachlorocyclopentadiene. Unpublished rep. Battelle Columbus
Lab. for U.S. Environ. Prot. Agency Health Res. Lab., Cincinnati, Ohio.
Bennett, T.B. 1977. Legal affidavit filed in State of Georgia, Clarke Co.,
June 15, 1977, concerning sampling of discharge at Velsicol Chemical Corp.,
Memphis, plant.
Bevenue, A. and A.Y. Yeo. 1969. Gas chromatographic characteristics of
chlordane. II. Observed compositional changes of the pesticide in aaueous
and non-aqueous environments. Jour. Chromatog. 42: 45.
Carter, M.R. 1977. Legal affidavit filed in State of Georgia, Fulton Co.,
dated June 14, 1977. Testimony concerning estimates of total daily dis-
charge of hex from Velsicol Chemical Corp. Memphis plant and calculations
of estimated hex concentration in Mississippi River resulting from said
discharge.
Equitable Environmental Health, Inc. 1976. Literature review of the health
and ecological effects of exposure to C-56 (Hexachlorocyclopentadiene).
Unpubl. rep. prepared for Hooker Chemical and Plastics Corp.
Harris, C.R. 1972. Behavior of dieldrin in soil: Laboratory studies on the
factors influencing biological activity. Jour. Econ. Entomol. 65: 8.
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Industrial Bio-Test Laboratories, Inc. 1977. Mutagenicity of PCL-HEX in-
corporated in the test medium tested against five strains of Salmonella
typhimurium and as a vslatilaie against tester strain TA-100. Unpubl. rep.
submitted to Velsicol Chemical Corp.
Ingle, L. 1953. The toxicity of chlordane vapor. Science. 118: 213.
Kirk, D.E. and D.F. Othmer. 1964. Kirk-Othmer Encyclopedia of Chemical
Technology. 2nd ed. Interscience Publishers, New York.
Lu, P.Y., et al. 1975. Evaluation of environmental distribution and fate
of hexachlorocyclopentadiene, chlordane, heptachlor, and heptachlor epoxide
in a laboratory model ecosystem. Jour. Agric. Food chem. 23: 967.
Marks, D.R. 1977. Description and data concerning monitoring of discharge
from Velsicol Chemical corp. Memphis plant to City of Memphis wastewater
collection system during January, 1977. Letter to Donald I. Mount, Direc-
tor, Environ. Res. Lab., U.S. Environ. Prot. Agency, dated February 3.
Metcalf, R.L. 1971. Model ecosystem for the evaluation of pesticides bio-
degrading and ecological magnification. Environ. Sci. Technol. 5: 708.
National Academy of Sciences. 1977. Kepone/Mirex/Hexachlorocyclopenta-
diene: An environmental assessment (final draft). Environ. Stud. Board.
Comm. Natl. Res. Counc.
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National Cancer Institute. 1977. Summary of data for chemical selection.
Internal working paper, Chemical Selection Working Group. U.S. Dept. Health
Edu. Welfare, Pub. Health Servu, Washington, D.C. (Unpubl.)
Reicke, C.E. 1977a. Effect of hexachlorocyclopentadiene on soil microbe
population. Unpubl. rep. submitted to Velsicol Chemical Corp., Chicago, 111.
Reicke, C.E. 1977b. Soil metabolism of 14C-hexachlorocyclopentadiene.
Unpubl. rep. submitted to Velsicol Chemical Corp., Chicago, 111.
Sanders, H.J. 1978. Flame retardants. Chem. Eng. News: April 24, 22.
Spehar, R.L. et al. 1977. A rapid assessment of the toxicity of three
chlorinated cyclodiene insecticide intermediates to fathead minnows. Off.
Res. Dev., Environ. Res. Lab., U.S. Environ. Prot. Agency, Duluth, Minn.
Swanson, D. 1976. Dishcarges from Hooker Chemical Company. Internal staff
rep. (unpubl.), Toxic Mater. Section, water Quality Div., Environ. Prot.
Bur. Mich. Dep. Nat. Resour.
Ungnade, H.E. and E.T. McBee. 1958. The chemistry of perchlorocyclopenta-
dienes and cyclopentadienes. Chem. Rev. 58: 240.
U.S. EPA. 1979. Water Related environmental fate of 129 priority pollut-
ants. EPA Contract No. 68-01-3852 U.S. Environ. Prot. Agency, Washington
D.C.
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Whitacre, D.M. 1978. Comments on document entitled, Review of the environ-
mental effects of pollutants. XI. Hexachlorocyclopentadiene, dated August 9.
Zavon, M.R. 1978. Letter to G.A. Lutz, Battelle Columbus Lab., dated
March 13.
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Aquatic Life Toxicology*
INTRODUCTION
Freshwater acute data are* available for several fish species and hexa-
chlorocyclopentadiene. An embryo-larval test has been conducted with the
fathead minnow. Results of tests with Daphnia magna indicate that it may be
more sensitive than the fish.
Acute tests with six saltwater species have been conducted and, with
the exception of a polychaete, these species were similarly sensitive to
this compound.
EFFECTS
Acute Toxicity
Two results are available for Daphnia magna with good reproducibility
between investigators; the 50 percent effect levels were 39 and 52 ug/1
(Table 1).
Henderson (1956) exposed the fathead minnow under three different con-
ditions using two dilution waters. One test water had a hardness of 40 mg/1
and pH of 7.4, and the second test water had a hardness of 400 mg/1 and pH
of 8.2. Two tests with hard water were conducted to evaluate the method
used to add the chemical to the dilution water. The latter comparison was
important since hexachlorocyclopentadiene is quite volatile and has an ex-
tremely low solubility in water. The chemical was added in a 0.01 percent
acetone solution or a 0.001 percent suspension of an emulsion prepared in a
blender. The effect of hardness, if any, was slight with 96-hour LC5Q
values of 104 ug/1 in soft water and 78 ug/1 in hard water (Table 1). The
*The reader is referred to the Guidelines for Deriving Water Quality
Criteria for the Protection of Aquatic Life and Its Uses in order to better
understand the following discussion and recommendation. The following
tables contain the appropriate data that were found in the literature, and
at the bottom of each table are calculations for deriving various measures
of toxicity as described in the Guidelines.
B-l
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test results comparing methods of addition were 78 and 59 ug/1 (Table 1) in-
dicating little difference. Spehar, et al. (1979) also determined a 96-hour
LCgg value for larvaU fathead minnows; this result, using flow-through
procedures and measured concentrations, was 7.0 ug/1 (Table 1). The differ-
ence between the data of Henderson (1956) and Spehar, et al. (1979) may be
due to differences in test methods or in relative sensitivity of different
life stages of the fathead minnow; the species acute value is 7.0 ug/1 since
there is only one flow-through test with measured concentrations. The chan-
nel catfish and bluegill 96-hour LC5Q values indicate that they are
similar to the fathead minnow in sensitivity to hexachlorocyclopentadiene
(Table 1).
Of the six saltwater species for which LC5Q values are available, the
polychaete was most resistant with a 96-hour LC5Q of 371 ug/1 (Table 1).
The other two invertebrate species were similar in sensitivity to the three
fish species with a range of LC50 values from 32 to 48 ug/1 for the five
species tested under static conditions. The mysid shrimp was also tested
under flow-through conditions and measured concentrations, giving a 96-hour
LC,jg of 7.0 wg/1. The related static result was 32 ug/1. This comparison
indicates that static procedures will probably underestimate the toxicity of
hexachlorocyclopentadiene.
Chronic Toxicity
The chronic value for the fathead minnow embryo-larval test by Spehar,
et al. (1979) is 5.2 ug/1 (Table 2). This concentration is not much lower
than the 96-hour LC_0 value (7.0 ug/1) for larval fathead minnows and re-
sults in an acute-chronic ratio of 1.3 (Table 2).
Plant Effects
No data are available on the effects of hexachlorocyclopentadlene on
freshwater or saltwater algae or plants.
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Residues
The bioconcentration factor for whole-body fathead minnows is 11 (Table
3) after a 30-day exposure (Spehar, et al. 1979). No Residue Limited Toxi-
cant Concentration can be determined since there is no permissible tissue
residue concentration available.
Miscellaneous
Applegate et al. (1957) exposed sea lamprey, rainbow trout, and blue-
gill to concentrations of hexachlorocyclopentadiene of 1,000 and 5,000 ug/l
(Table 4). Death or distress was observed in one-half to one hour. The 30-
day LCgQ value for the fathead minnow (Spehar, et al. 1979) is 6.7 ug/l
which result is only slightly lower than the 96-hour LC5Q value of 7.0
ug/l determined by the same investigators.
Summary
Hexachlorocyclopentadiene is very toxic to freshwater organisms. Under
static conditions, 50 percent effect concentrations for Daphnia magna and
three fish species were in the range of 39 to 180 yg/1. A comparison of
static and flow-through conditions indicates that the latter yields signifi-
cantly lower lethal values. The chronic value for the fathead minnow was
5.2 ug/l, a concentration only slightly below a lethal concentration for
that species. Residues of hexachlorocyclopentadiene do not appear to be a
problem with a bioconcentration factor in fish of 11. The 30-day LC5Q
value for the fathead minnow was 6.7 ug/l, a concentration only slightly
lower than the flow-through 96-hour LCgQ of 7.0 wg/1.
The saltwater data base is more limited, with 96-hour LC5Q values for
three invertebrate and three fish species obtained under static conditions
in the range of 32 to 48 ug/l ''for all species except the polychaete for
B-3
-------
which the LC5Q value was 371 ug/1. As with the fathead minnow, the flow-
through LC5Q for the mysid shrimp was significantly lower than the static
test result with the same species. No other data are available for salt-
water organisms.
CRITERIA
The available data for hexachlorocyclopentadiene indicate that acute
and chronic toxicity to freshwater aquatic life occur at concentrations as
low as 7.0 and 5.2 ug/1, respectively, and would occur at lower
concentrations among species that are more sensitive than those tested.
The available data for hexachlorocyclopentadiene indicate that acute
toxicity to saltwater aquatic life occurs at concentrations as low as 7.0
ug/1 and would occur at lower concentrations among species that are more
sensitive than those tested. No data are available concerning the chronic
toxicity of hexachlorocyclopentadiene sensitive saltwater aquatic life.
B-4
-------
Table 1. Acute values for hexachlorocyclopentadlana
W
en
Species
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla magna
Fathead minnow (larva),
Plmephales promelas
Fathead minnow,
Plmephales promelas
Fathead minnow,
Plmephales promelas
Fathead minnow,
Plmephales promelas
Fathead minnow,
Plmephales promelas
Channel catfish,
Ictalurus punctatus
Blueglll,
Lepomls macrochlrus
Polychaete,
Neanthes arenaceodentata
Mysld shrimp,
Mysldopsls bah la
My si d shrimp,
Mysldopsls bah I a
Grass shrimp.
Method*
s.
s.
FT,
s.
s.
s.
s.
s,
s.
s,
s.
FT.
s.
U
u
M
U
U
U
U
U
U
U
U
M
U
Species Mean
LC50/EC50 Acute Value
(Ufl/l) (yg/l)
FRESHWATER SPECIES
39
52 45
7.0
104
78
59
180 7.0
97 97
130 130
SALTWATER SPECIES
371 371
32
7.0 7.0
42 42
Reference
EG & G, Bionomics,
1977
Union Carbide Environ'
mental Services, 1977
Spehar, et al.
1979
Henderson, 1956
Henderson, 1956
Henderson, 1956
EG & G, Bionomics,
1977
EG & G, Bionomics,
1977
EG & G, Bionomics,
1977
U.S. EPA, 1980
U.S. EPA, 1980
U.S. EPA, 1980
U.S. EPA, 1980
Palaemonetes puglo
-------
Table 1. (Continued)
LC50/EC50
Species Method*
-------
Table 2. Chronic values for hexachlorocyclopentadiene
(Spehar, at al. 1979)
Chronic
Limits Value
Method* (U9/D (iig/l)
Fathead minnow,
Plmephales promelas
FRESHWATER SPECIES
ELS 3.7-7.3
5.2
* ELS = early life stage
Acute-Chronic Ratio
CO
I
-J
Spec 1 es
Fathead minnow.
Plmephales promelas
Acute
Value
(ug/D
7.0**
Chronic
Value
-------
Table 3. Residues for hexachlorocyclopentadlene (Spehar, et al. 1979)
Bloconcentratlon Duration
Tissue Factor (days)
FRESHWATER SPECIES
Fathead minnow (juvenile), whole body 11 30
Plmephales promelas
CO
I
oo
-------
Table 4. Other data for hexachlorocyclopentadlene
03
I
vo
Species
Sea lamprey (larva),
Petromyzon marlnus
Sea lamprey (larva),
Petromyzon marlnus
Rainbow trout
(fIngerlIng),
Salmo galrdnerl
Rainbow trout
(fIngerlIng),
Salmo galrdnerl
Fathead minnow (larva),
Plmephales promelas
Blueglll (fIngerlIng),
Lepomls macrochlrus
Blueglll (fIngerlIng),
Lepomls macrochlrus
Duration Effect
FRESHWATER SPECIES
24 hrs
Death In I hr
ResiilT
(U9/D
5,000
24 hrs Distress In 1/2 hr 1,000
24 hrs Death In 1/2 hr 5,000
24 hrs Death In I hr
Reference
App legate, et a I. 1957
Applegate, et al. 1957
App legate, et al. 1957
1,000 App legate, et al. 1957
30 days LC50 6.7 Spehar, et al. 1979
24 hrs Death In 1/2 hr 5,000 Applegate, et al. 1957
24 hrs Distress In 1/2 hr 1,000 Applegate, et al. 1957
-------
REFERENCES
Applegate, V.C., et al._i 1957. Toxicity of 4,346 chemicals to larval lam-
t
preys and fishes. U.S. Fish. Wild. Serv. Spec. Rep. — Fish. No. 207.
Washington, D.C., U.S. Dep. of Inter.
EG a- 6, Bionomics. 1977. Acute toxicity of hexachlorocyclopentadiene to
bluegill (Lepomis macrochirus), channel catfish (Ictalurus punctatus), fat-
head minnow (Pimephales promelas) and the water flea (Daphnia magna). Toxi-
city Test Report submitted to Velsicol Chemical Corporation, Chicago,
Illinois.
Henderson, D. 1956. Bioassay investigations for International Joint Com-
mission. Hooker Electrochemical Co., Niagara Falls, N.Y. U.S. Dep. of
Health, Educ., and Welfare, Robert A. Taft Sanitary Engineering Center, Cin-
cinnati, Ohio.
Spehar, R.L., et al. 1979. Toxicity and bioaccumulation of hexachloro-
cyclopentadiene, hexachloronorbornadiene and heptachloronorbornene in larval
and early juvenile fathead minnows, Pimephales promelas. Bull. Environ.
Contam. Toxicol. 21: 576.
Union Carbide Environmental Services. 1977. The acute toxicity of hexa-
chlorocyclopentadiene to the water flea, Daphnia magna Straus. Prepared for
Velsicol Chemical Corporation, Chicago, Illinois.
U.S. EPA. 1980. Unpublished laboratory data. Environmental Research
Laboratory-Gulf Breeze.
B-10
-------
Mammalian Toxicology and Human Health Effects
EXPOSURE
Ingestion from Water
Very little is known regarding potential hex exposures through
ingestion of contaminated food or water. Hexachlorocyclopentadiene
(Hex) has been detected in specific bodies of water near points of
industrial discharges. Except for such source-directed sampling,
it appears that there is little information on hex concentrations
in surface waters. Hex is usually not detectable in water samples.
Due to its low solubility and tendency to volatilize, one would not
expect it to remain in flowing water. Moreover, there are no data
on hex levels in drinking water or the extent to which hex in raw
(untreated) water would be passed through the water treatment pro-
cess to human consumers.
Ingestion from Food
Hex has been identified in a few samples of fish taken from
waters near the Hooker plant in Michigan (Spehar, et al. 1977).
Frequently, however, hex residues have not been detected in edible
fish deliberately exposed to hex in laboratory experiments. Ac-
cording to the same investigator, the inability to recover hex in
fish samples probably results from losses by vaporization during
sample extraction. No reports concerning hex contamination of
other foods could be located (Spehar, et al. 1977).
A bioconcentration factor (BCF) relates the concentration of a
chemical in aquatic animals to the concentration in the water in
which they live. The steady-state BCFs for a lipid soluble com-
C-l
-------
pound in the tissues of various aquatic animals seem to be propor-
tional to the percent lipid in the tissue. Thus, the per capita
ingestion of a lipid^-soluble chemical can be estimated from the per
capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and shellfish, and a steady-state BCF
for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States were analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the per capita con-
sumption of freshwater and estuarine fish and shellfish in the
United States is 6.5 g/day (Stephan, 1980). In addition, these
data were used with data on the fat content of the edible portion of
the same species to estimate that the weighted average percent
lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
A measured steady-state bioconcentration factor of 11 was ob-
tained for hexachlorocyclopentadiene using fathead minnows
(Spehar, et al. 1979). Similar fathead minnows contained an aver-
age of 7.6 percent lipids (Veith, 1980). An adjustment factor of
3.0/7.6 = 0.395 can be used to adjust the measured BCF from the 7.6
percent lipids of the fathead minnow to the 3.0 percent lipids that
is the weighted average for consumed fish and shellfish. Thus, the
weighted average bioconcentration factor for hexachlorocyclo-
pentadiene and the edible portion of all freshwater and estuarine
aquatic organisms consumed by Americans is calculated to be 11 x
0.395 = 4.34.
C-2
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Inhalation
The heaviest and most chronic exposure to hex undoubtedly
occurs among persons epgaged directly in the manufacture of hex and
among production workers fabricating hex-containing products. Al-
though several cohorts of hex-exposed workers can be specifically
identified (employees of Hooker Chemicals and Plastics, Michigan
and Niagara Falls plants; Velsicol Chemical Corporation, Memphis
plant; Shell Chemical Company, Denver and Pernis, Netherlands,
plants; an Israeli company, Makhteshim; and the Hooker plant at
Genk, Belgium), there have been no reports of epidemiologic studies
of these workers. Hooker Chemicals and Plastics Corporation, a
manufacturer of hex, reported that they are presently conducting a
mortality study of chronically exposed hex workers, but the study
is in its initial stages and very likely will not be completed
until 1980 (Zavon, 1978, personal communication). Inhalation of
hex is the primary mode of occupational exposure. Accidental
spills and illegal discharges of hex represent the primary mode of
acute human exposure (e.g., the Louisville incident). Inhalation
appears to be the most important mode of exposure in these cases as
well.
Dermal
According to Hooker Chemical and Plastic Corporation's Mate-
rial Safety Data Sheet, hex is readily absorbed through the skin.
Prolonged or repeated contact can lead to burns and manifestations
of systemic toxicity not unlike those caused by inhalation. The
hazards of skin contact are well recognized and industrial workers
are provided with impervious clothing to prevent dermal contact
C-3
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(Hooker, 1972). Thus, dermal exposure should not be anticipated
among workers familiar with hex. Persons outside the chemical in-
dustry can be exposed to hazardous contacts as a result of acci-
dental spills or improper disposal of hex.
PHARMACOKINETICS
Two studies which address the pharmacokinetics of hex could be
located (Mehendale, 1977; Dorough, 1979). The Mehendale study
focuses upon the disposition of hex within the body and its modes
of elimination, and the Dorough study reported accumulation, dis-
tribution, and elimination of hex in mice and rats.
In the Mehendale (1977) study, radiolabeled hexachlorocyclo-
14
pentadiene ( C-hex) was administered by oral intubation to four
male Sprague-Dawley rats in order to examine absorption, metabo-
lism, and excretion of the compound following a single oral dose.
After dosing with C-hex (5 pmoles, 1 pCi per animal) , the rats
were maintained in metabolism cages for seven days, during which
daily urine and fecal samples were collected. After seven days,
the animals were sacrificed and the major organs were removed and
radioassayed.
Urine and powdered fecal samples were radioassayed for total
14
C. An average of approximately 33 percent of the total dose was
excreted in the urine after seven days. About 87 percent of that
(approximately 28.7 percent of total dose) was eliminated during
the first 24 hours after the administration of the compound. Fecal
excretion accounted for 10 percent of the total dose; nearly 60
percent of the 7-day fecal excretion occurred during the first day.
C-4
-------
Beyond the third day after treatment, only trace amounts of
the hex-derived C were eliminated in the feces. Tissues retained
only trace amounts of hex after seven days. For example, the kid-
ney retained only about 0.5 percent of the total dose and the liver
less than 0.5 percent. Other organs and tissues — fat, lung, mus-
cle, blood, etc. — contained even less of the radiolabel. Such
findings suggest that at least half of the administered hex was
eliminated by routes other than urine and feces. The author felt
that the respiratory tract is probably the major route of excre-
tion.
The nature of the radioactivity excreted in the urine was ex-
amined searching for possible metabolites. It was found that about
70 percent of the radioactivity in the urine was extractable using
a hexane:isopropanol (9:1) mixture. The organic solvent was con-
centrated, applied to thin-layer chromatography (TLC) plates, and
developed in three solvent systems. The radioactive spots were
visualized by auto-radiography on medical x-ray film. The results
suggested the presence of at least four metabolites; however, at
the time of this writing they had not been identified and charac-
terized.
Disposition and biliary excretion of C-hex was studied by
14
injection of approximately 1 yCi (5 umole) of C-hex into the
femoral vein of anesthetized rats. Timed samples of blood and bile
were collected for one hour from the femoral artery and common bile
duct which had been cannulated prior to dosing. Approximately 9
percent of the administered dose was excreted in the bile in one
hour. Because this quantity is equivalent to that excreted in the
C-5
-------
faces over seven days, enterohepatic circulation of this compound
is probable. The nature of the compound present in the bile is not
yet known.
At the end of the above experiments, the animals were sacri-
ficed and the liver and kidneys were removed. Tissue homogenates
from these organs were radioassayed and the distribution of the
radioactivity among the various subcellular fractions was examined
by assaying the various centrifugation fractions. Kidney cytosol
accounted for 93 percent of the radioactivity in the total kidney
homogenate. This behavior is consistent with rapid urinary excre-
tion. Similarly, 68 percent of the radioactivity in the liver
homogenate was associated with the liver cytosol fraction, once
again consistent with rapid excretion.
Pre-exposure of some of the rats to hex (50 mg/kg/day) for
three days prior to the experiment did not affect blood decay
curves and biliary excretion; however, an increased concentration
14
in the kidneys after a single challenge with C-hex was observed.
Dorough (1979) reported a pharmacokinetic investigation of hex
completed under contract to Velsicol Chemical Corporation.
In a single oral dose study, 14C-labeled-hex was given by gav-
age to Sprague-Dawley rats and albino mice of both sexes in two
dosages, 2.5 mg/kg and 25 mg/kg. Animals were kept in metabolism
cages and feces and urine were collected separately. Animals were
killed on days one, three and seven post-treatment. There was no
appreciable difference in excretion patterns between species or
sex, and the major route of excretion was through the feces with
83.4 percent of the 2.5 mg/kg dose and 85.5 percent of the 25 mg/kg
C-6
-------
dose excreted by the third day after treatment. Rats showed maxi-
14
mum C-residues in the kidney/ whereas in mice, maximum residue
levels were found in the liver.
In a continuous feeding study, male and female Sprague-Dawley
rats and male and female albino mice were placed on diets contain-
ing 1, 5, or 25 ppm hex for a maximum of 30 days. Animals were
killed at intervals during and after initiation of the study. Ex-
cretion patterns were the same for rats and mice, and no sex dif-
ferences were noted. The major route of excretion was through the
feces.
At all dose levels in all animals the kidney, liver, and adi-
pose tissue contained the highest residue levels; and apparent
equilibration had been attained after 15 days of feeding, and a
positive correlation was observed between the levels of hex in the
diet and in the tissues.
It appears that results of this study do not agree closely
with the Mehendale study. The Dorough study shows hex to be elimi-
nated from mammals (mice and rats) mainly by the fecal route and
with no more than about 15 percent being eliminated in urine. Fur-
ther, these studies do not indicate any significant amounts of pul-
monary elimination of hex or its metabolites. Whitacre (1978) be-
lieves that the poor recoveries in feces in the Mehendale study may
be the result of volatility of hex or its metabolites before remov-
al for analysis. Losses during sample preparation undoubtedly fur-
ther complicate the analysis of fecal matter.
C-7
1
-------
EFFECTS
Acute, Subacute, and Chronic Toxicity
The classic studies of hex toxicity to mammals were conducted
in the mid 1950's by Treon, et al. (1955). This series of investi-
gations reported on both acute and subacute toxicity of hex to var-
ious species of mammals under a variety of exposure regimens.
Oral, dermal, and inhalation modes of exposure were included in
Treon's experiments. Mammalian toxicity studies subsequent to the
1950's could not be located in the open literature, probably due to
the rather low profile of hex relative to other pesticide chemi-
cals. More recent, proprietary studies of the oral and dermal
toxicity have now become available. In general, these findings
agree remarkably well with those of Treon. It is most unfortunate
that no truly long-term (i.e., longer than six months) studies of
chronic effects have been conducted. Until data on the potential
effects of long-term, chronic exposure (especially carcinogeni-
city) becomes available, any recommendations regarding environ-
mental criteria must be regarded as tentative.
Acute toxicity of hex was determined by Treon, et al. (1955)
by administering dosages of 180, 280, 340, 420, 520, 620, 940,
1,400, and 2,100 mg/kg of hex in peanut oil directly into the stom-
achs of several groups of rabbits and rats. The data on rabbits
indicate that the median lethal oral dose (LD5Q) administered as
described above, lies in the range between 420 and 620 mg/kg of
body weight.
Rats showed variation in minimum lethal dose depending on sex.
Male rats were somewhat more sensitive in that the lethal dose was
C-8
-------
somewhat less than 280 mg/kg body weight, whereas for females the
dosage causing death was greater than 280 mg/kg. The LD5Q for male
rats was determined to be 505 mg/kg with 95 percent confidence
limits of 387-623 mg/kg.
Kommineni (1978) conducted a study which focused upon gross
and histopathological effects at the possible routes of entry and
elimination of hex.
In the Kommineni study, a total of 10 female rats were exposed
to 0, 50, 100, 150, 200, and 300 mg/kg of hex by gavage. All ani-
mals were sacrificed 24 hours pjost-treatment. The rats were
necropsied and lungs, liver, spleen, kidneys, adrenals, heart,
stomach, and intestines were saved for histopathology evaluation.
Gross pathology of the rats exposed to 200 and 300 mg/kg re-
vealed brown discoloration around the nostrils and anus of the
rats. The urinary bladders of two of the four rats contained brown
fluid. Subserosal emphysema of the nonglandular stomach was evi-
dent in one animal. On histopathologic examination, the lungs
showed atelectasis with moderate thickening of the alveolar walls.
The alveolar walls contained moderate numbers of macrophages and
neutrophils. Some bronchi contained denuded epithelium. No edema
was present in the lungs. Rats receiving lower dosages showed
similar, but milder, changes. The stomachs of rats receiving dos-
ages of 200 or 300 mg/kg showed coagulative necrosis of the gastric
squamous epithelium. The submucosa of the nonglandular part of the
stomach showed mild neutrophilic infiltration. The supporting
structures of the stomach (submucosa, submuscularis, muscular is)
showed moderate edema. Epithelium of the glandular part of the
C-9
-------
stomach showed no treatment-related changes. Animals receiving
lower doses showed similar changes in the stomach. Ulcers of the
nonglandular portion of the stomach were seen in several of the
animals. At all dosages, the other organs were unremarkable.
The author commented that these morphological changes indicate
that hex is absorbed through the squamous epithelium of the non-
glandular part of the stomach and that the major route of elimina-
tion of hex is through the lungs.
The International Research and Development Corporation (IRDC,
1972) conducted similar studies of the acute oral toxicity of hex.
Twenty-five albino rats of each sex were given hex dissolved in
corn oil at dosages of 315, 500, 794, 1,250, and 1,984 mg/kg. Five
rats of each sex were used at each dosage level. An LD5Q of 530
mg/kg was determined for female rats and 630 for male rats. The
combined oral LD5Q for both sexes was determined to be 584 mg/kg.
Note that this is the reverse of the sex difference reported by
Treon, et al. (1955). Naishstein and Lisovskaya (1965) reported a
LD50 of 600 mg/kg for white rats. This value is comparable to the
upper part of the range (420-620 mg/kg) reported by Treon, et al.
(1955). Thus, the true LD50 is probably about 600 mg/kg.
In this series of experiments, 93.3 percent hexachlorocyclo-
pentadiene was applied to the intact skin of rabbits using the
technique of Draize et al., described by Treon et al. (1955). It
was determined that the lethal dosage lies between 430 and 630
mg/kg body weight. Such a finding is notable in that hex appears to
be just as toxic via dermal application as by ingestion.
C-10
-------
Kommineni (1978) , painted four male guinea pigs on the skin
(site unspecified) with hex at dosages of 0, 300, 600, and 1,200
rag/kg and sacrificed 24 hours after the exposure. All animals were
necropsied and the lungs, liver, pancreas, kidneys, adrenals, uri-
nary bladder, heart, skin, stomach, and intestines were saved for
histopathologic evaluation.
On gross pathology, subcutaneous edema was seen extending from
the inguinal area to the sternum. At the lowest dosage, the lungs
were highly expanded and showed rib impressions on the parietal
surface. Similar but more severe changes were seen in the animal
receiving 600 mg/kg. The animal painted with 1,200 mg/kg expired
prior to sacrifice; the trachea was filled with frothy fluid. His-
topathologic examination of the lungs revealed atelectasis with
thickened alveolar walls containing moderate numbers of macrophages
and neutrophils. Intense congestion of all pulmonary blood vessels
and occasional alveolar edema was seen in the animal receiving the
1,200 mg/kg dose. In the skin, moderate to marked edema disrupted
the collagen bundles. Focal pockets of neutrophils were seen in
the edematous dermis. Edema extended throughout the thickness of
the adipose tissue layer. One animal showed partial thrombosis of
medium size veins situated deep in the dermis. The skin appendages
were normal.
More recently, the irritant properties of hex were examined in
a study conducted by IRDC (1972). These tests were commissioned by
Velsicol Chemical Corporation in accordance with the regulations of
the Federal Hazardous Substances Act.
C-ll
-------
IRDC (1972) reported the results of an investigation of acute
dermal toxicity of hex to rabbits. Four male and four female New
Zealand White rabbits -were used in this test. The hair was removed
from the back of each rabbit with electric clippers. Two male and
two female rabbits were used at each of two dosage levels. The test
compound was applied in a single administration to the back of each
rabbit at a dosage of 200 or 2,000 mg/kg body weight. The area of
application was wrapped with a gauze bandage and occluded with
Saran Wrap. Twenty-four hours later, the bandages were removed and
the backs were washed with water. The rabbits were observed for
mortality for a period of 14 days.
All of the animals which received 2,000 mg/kg dosage died
within 24 hours after application of the compound. At the 200
mg/kg dosage, both male rabbits died but both female rabbits sur-
vived although they both exhibited weight loss over the 14-day
period. The male rabbits that died showed weight loss also. In
addition, cachexia, marked dermal irritation, and hypoactivity was
observed. Skin at the site of application turned purple within a
few hours after hex application. Based on these results, hex was
concluded to be "a highly toxic material by the dermal route of ex-
posure" in accordance with the criteria established under the Fed-
eral Hazardous Substances Act.
Treon, et al. (1955) exposed various animal species to vapors
formed by bubbling a stream of air through liquid hex contained in
a bubbling tower. This air was then mixed with clean air to achieve
the desired concentration. The stream of air, conditioned with re-
spect to temperature, dust content, and humidity, was then passed
C-12
-------
into a plywood exposure chamber in which the test animals were con-
fined. A series of hex concentrations in the air in the exposure
chamber were used; these varied from 0.15 to 73.6 ppm. Test spe-
cies were guinea pigs, rats, mice, and rabbits.
The authors reported that hex vapor was very toxic to all four
species of animals. Exposure to the concentration of 13.0 ppm (an
intermediate level in this experiment) for 15 minutes produced
fatalities in all species except guinea pigs. Of the four species,
rabbits appeared to be the most susceptible. Mice, rats, and
guinea pigs followed in order of decreasing susceptibility.
Table 1 depicts results of the inhalation experiments. The values
tabulated correspond to the concentration in ppm which: (1) per-
mitted all animals to survive; (2) killed 50 percent of the ani-
mals; and (3) killed 100 percent of the animals.
Animals of the following species died regularly when exposed
to hex vapors at the following concentrations and durations: rab-
bits - 1.5 ppm for seven hours; mice - 1.4 ppm for two 7-hour
periods; rats - 1.0 ppm for five 7-hour periods or 3.2 ppm for two
7-hour periods; and guinea pigs - 3.2 ppm for two 7-hour periods.
IRDC (1972) also reported the results of acute inhalation ex-
periments in rats. The test animals were exposed to atmospheric
concentrations of approximately 176.2 and 17,624 ppm of the test
compound for four hours. Ten rats were tested at each dosage
level. Due to the extremely high dosages employed, little informa-
tion could be derived from the study. No justification of the
choice of dosages was given. All of the animals receiving the test
compound at either exposure level died within 48 hours. All rats
C-13
-------
TABLE 1
Dose Response Data: Inhalation of Hex Vapors*
Species of Animal
Guinea pigs. . .
Rats . . .
Mice. . .
Rabbits. . .
Fatalities,
Percent
0
50
100
0
50
100
0
40
100
0
67
100
Hex Concentration (inppm) Lethal
to % of Test Animals Indicated
1-Hour 3ij-Hour 7-Hour
Exposure Exposure Exposure
7.2
13.8
20. Oa
3.1
7.2
20. Oa
1.4
7.2
13.8
1.4
3.1
7.2
3.1
7.1
12.4
1.4
3.1
7.1
1.4d
3.1e
7.1
—
6.4
7.1
1.5
3.2
6.7
1.5b
3.2C
6.7
—
1.5e
3.2
—
—
7.5
*Source: Treon, et al. 1955
aDuration of exposure was 1.25 hours
tJ <•« ^ - — .. j_ ._. £ .» «• >«k» « -P-I >3 •! f\.A
325 percent of group died
375 percent of group died
320 percent of group died
380 percent of group died
C-14
-------
at the 17,624 ppm dosage level died during the 4-hour exposure
period. At the 176.2 ppm atmospheric concentration, one rat died
during the exposure period, eight more were dead within 24 hours,
and the remaining rat died on the second day of observation.
Signs seen during the exposure period included eye squint,
dyspnea, cyanosis, salivation, lacrimation, and nasal discharge.
Gross necropsy showed gray coloration of the skin, severe hemor-
rhage of the lungs, and hydrothorax among rats exposed to 17,624
ppm. Rats exposed to 176.2 ppm revealed congestion of the lungs in
all cases.
Based on these results, the investigators concluded that hex
is highly toxic material by the inhalation route of administration.
Table 2 summarizes the results of acute toxicity studies of hex.
To date, there has not been a satisfactory study of subacute
or chronic oral toxicity of hex. One portion of the Treon, et al.
(1955) study attempted to examine subacute/chronic oral toxicity
but reported that dosages of 180-2,100 mg/kg were fatal within such
a short period of time that the investigators were unable to estab-
lish an oral dosage which could be tolerated without mortality over
an extended period. Similarly, Naishstein and Lisovskaya (1965)
reported that oral administration as little as 20 mg/kg for six
months was fatal to 20 percent of white rats.
Treon, et al. (1955) examined effects of sublethal concentra-
tions of hex applied to the skin of rabbits and monkeys. In rab-
bits, dosages as low as 250 mg/kg induced extreme irritation,
purplish-black discoloration of the skin and subcutaneous edema.
Although the skin lesions healed eventually, damage to the skin in
C-15
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TABLE 2
Acute Toxicity of Hexachlorocyclopentadiene
By Various Modes of Exposure*
Oral administration'
Animal
Rabbits
Rats
Ratsd
Females
Males
LD
50
420-620 mg/kg
505 mg/kg
530 mg/kg
630 mg/kg
Minimum Lethal Dose
Rats
Males
Females
280 mg/kg
250 mg/kg
Dermal application*3
LD
50
Rabbits
430-630 mg/kg
Inhalation0
LD50 - (dosage expressed as vapor
concentration, ppm)
Guinea pig
Rats
13.8 ppm
7.2 ppm
*Source: Treon, et al. 1955
aHex dissolved in peanut oil, administered by gavage
b93.3 percent hex solution in Ultrasene, applied to intact skin
for 24 hours
°LD5Qs based on 1-hour vapor exposure
dBased on data reported by International Research and Development
Corp. (1972). Hex dissolved in corn oil
C-16
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the area of application persisted for many days and the damage
varied in severity and extent with the amount (dosage) of the ma-
terial applied.
A slightly different procedure was employed in the cutaneous
exposures of the monkeys. In this case, a series of hex concentra-
tions (0.001, 0.01, 0.1, 1.0, and 10.0 percent) dissolved in Ultra-
sene were applied to five sites of the abdominal skin. Dosage of
each of the solutions was 0.01 ml. No irritation or other changes
were noted; however, when 0.05 ml of the 10 percent solution was
applied to the back of a monkey for three consecutive days, the
skin became severely irritated and necrotic. Subsequent experi-
ments used more concentrated solutions (20, 40, 60, and 90 percent)
which were applied (dosage of 0.05 ml) on separate areas of the
monkeys' backs. At all concentrations there was discoloration of
the skin, ranging from very light to dark tan as the concentration
increased. The discoloration was followed by swelling which varied
from slight to severe, again depending on concentration. The
highest concentration caused cracking, oozing, and serious dis-
charge from the treated areas; intermediate concentrations produced
hardening and swelling of the skin.
In guinea pigs, application of solutions containing 0.01,
0.10, 1.0, and 10 percent hex caused no alterations of the skin,
but more concentrated solutions (40, 60, and 90 percent) resulted
in discoloration, hardening, and necrosis of the skin at the appli-
cation site. Based on these tests, it appears that the threshold
concentration at which hex in Ultrasene induces irritation of the
C-17
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intact skin lies between 10 and 40 percent for both monkeys and
guinea pigs.
Hex was tested for eye irritancy by instilling 0.1 ml of the
.*
"test compound" (which was presumably undiluted liquid hex) into
the eyes of New Zealand White rabbits (IRDC, 1972). The test mate-
rial was placed into the conjunctival sac of the right eye of each
rabbit; the left eye served as an untreated control. Corneal dam-
age was evaluated by instillation of sodium fluorescein into the
eye, followed by examination of the corneal surface for evidence of
damage under ultraviolet light. A graded scale was used to quanti-
fy the extent and severity of damage. The eyes of the rabbits were
checked for corneal lesions at intervals (at 1, 24, 48, and 72
hours post-exposure and at 7, 14, and 21 days post-exposure). Ex-
aminations at 14 and 21 days were precluded by the deaths of all of
the rabbits on or before the ninth day of the observation period.
IRDC investigators attributed the deaths to the effects of the test
compound, but unfortunately did not conduct postmortem examinations
to rule out other possible causes of death.
Based on the severity of the ocular lesions produced in the
rabbits, hex was concluded to be "an extreme irritant and probable
corrosive substance" in the 5-minute test and "an extreme irritant
and corrosive substance" in the 24-hour wash test (IRDC, 1972).
These classifications are set in accordance with standards set
under The Federal Hazardous Substances Act, specifically Part 191,
Hazardous Substances Test for Eye Irritants, Food and Drug Admini-
stration.
C-18
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When mice, rats, rabbits, and guinea pigs were exposed to 0.34
ppm in air for 7 hours a day for 5 days per week, none of the mice
or rats survived mor-e than 20 such exposures (Treon, et al. 1955).
Two-thirds of the rabbits had died by the end of the twenty-fifth
period; however, the guinea pigs survived through 30 periods. At
0.15 ppm, some animals from all four species survived 150, 7-hour
exposures over a period of 216 days. Eight percent of the mice did
not survive the prolonged intermittent exposure. Details of these
findings are discussed under the heading "chronic toxicity."
In the Treon, et al. (1955) study, rabbits and rats given
various dosages of hex ranging from 180-2,100 mg/kg tended not to
survive long enough at these dosages to provide acceptable data on
chronic oral toxicity. Consequently, these investigators were un-
able to establish an oral dosage which could be tolerated (e.g.,
without mortality) over an extended period of time.
Studies in the Soviet Union reported by Naishstein and Lisov-
skaya (1965) appear to provide the only source of information on
the effects of long-term, low-dose exposure to hex. Daily adminis-
tration of 1/30 of the median lethal dose (20 mg/kg) for six months
killed only 2 animals out of 10, even though the cumulative dose
received was 1.5 times the acute LD10Q, and six times the LD5Q. Al-
though some changes were noted in the weight coefficients of in-
ternal organs of the animals, the authors judged the cumulative ef-
fects of hex to be weak. No observations of neoplasms or other ab-
normalities were reported.
C-19
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Undiluted 93.3 percent hex solution in concentrations of 430
mg/kg, 610 mg/kg, 1,020 mg/kg, 2,130 mg/kg, and 6,130 mg/kg, when
applied to the skin of rabbits, was frequently fatal within a few
hours. Six rabbits who survived for 7-21 days after application of
hex were killed and autopsied. Degenerative changes were seen in
the brain, liver, kidneys, and adrenal glands of these animals in
addition to chronic skin inflammation, acanthosis, hyperkeratosis,
and epilation. Visceral lesions due to dermal hex application re-
ported by Treon, et al. (1955) are described in the section on
toxic symptoms and pathological effects.
Naishstein and Lisovskaya (1965) also investigated the effects
of multiple, low-dose dermal exposures to hex. These experiments
consisted of applying 0.5-0.6 ml of a concentration of 20 ppm hex
in aqueous solution to the shaved skin of rabbits daily for a
period of 10 days. No differences were detected between the skin
of the experimental animals and that of the controls.
Treon, et al. (1955) reported that dosages of less than 10
percent hex appeared to be tolerated without irritative effects in
monkeys and probably also in guinea pigs. Unfortunately, neither
investigation continued the low-dose regimen for a sufficient
period to observe chronic effects.
Treon, et al. (1955) exposed guinea pigs to hex vapors at a
concentration of 0.34 ppm hex for seven hours per day, five days a
week. All of them survived until they reached 30 periods of expo-
sure in six weeks. Rats and mice exposed to this concentration
survived only five periods of exposure; however, survival of the
C-20
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rabbits was intermediate; two-thirds had died before the end of the
fifth week (25 exposure periods).
A lower concentration, 0.15 ppm hex, was tolerated by guinea
pigs, rabbits, and rats throughout 150, 7-hour periods of exposure
extending over a period of approximately seven months. Four of
five'mice died within this period. Although guinea pigs, rabbits,
and rats appeared to grow normally during this period, slight
degenerative changes were observed in the livers and kidneys of
these animals. These changes are discussed in the following
section.
Rats and rabbits exposed to hex in the Treon, et al. (1955)
acute toxicity study exhibited diarrhea, lethargy, and retarded
respiration. The odor of hex could also be detected in the feces of
these animals and on their bodies, presumably from fecal contamina-
tion. Rabbits which died following exposure to moderately high
doses of hex (180-2,100 mg/kg hex in corn oil) showed diffuse de-
generative changes in the epithelium of the renal tubules. As in
the study of Kommineni (1978), the lungs of these animals were con-
gested and edematous. The same types of degenerative changes were
also noted in the rats. In addition, some of the rats showed acute
necrotic gastritis. Animals which survived the oral tests and were
later sacrificed exhibited residual degenerative changes of the
type described above, suggesting that the pathological changes are
persistent. The severity of the lesions was diminished however,
with increasing length of the post-exposure survival interval.
A 90-day subacute oral toxicity study in rats conducted for
Hooker Chemical and Plastics Corporation by Industrial Bio-Test
C-21
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Laboratories (IBT) was reported by Equitable Environmental Health
(1976) but the data from this study were not made available for
review.
Naishstein and Lisovskaya (1965) reported results of a chronic
oral toxicity experiment on 90 white rats. For a period of six
months rats were given daily peroral doses of 0.002, 0.0002, and
0.00002 mg/kg (0.04, 0.004, and 0.0004 mg/1) in aqueous solution.
The first dose was 30 times greater than the threshold concentra-
tion with respect to aftertaste and smell (0.0013 mg/1); the second
dose corresponded to the practical limit of detection by smell, and
the third dose was 10 percent of the second. No deviations were
observed in the behavior of the rats or in their weights throughout
the 6-month experimental period. Likewise, no significant changes
were seen in hemoglobin, red blood cells, white blood cells, or
peripheral reticulocyte counts in the experimental groups as
opposed to the controls. In animals receiving the highest dose,
0.002 mg/kg, neutropenia and a tendency toward lymphocytosis were
reported. The peripheral blood of animals receiving the two lower
dosages did not show any alterations relative to controls. The
authors concluded that daily peroral administration of doses of
0.0002 and 0.00002 mg/kg (0.004 and 0.0004 mg/1 in aqueous solu-
tion) caused no changes in peripheral blood cells, ascorbic acid
content, conditioned reflexes, or histologic structure of the
organs. Based on these tests and the threshold level for organo-
leptic noxious effects (smell and aftertaste in water), Naishstein
and Lisovskaya (1965) recommended a maximum permissible concentra-
tion of 0.001 mg/1 hex in water.
C-22
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Treon, et al. (1955) showed that application of very low dos-
ages of hex (0.25 mg/kg) to the skin of rabbits was extremely irri-
tating and induced local discoloration and edema. The skin became
hard, encrusted, and fissured several days after application. The
extent of the local damage varied directly with the size of the
dose applied. At autopsy rabbits exhibited visceral lesions simi-
lar in appearance to those seen after oral administration of hex.
Again, diffuse degenerative changes were seen in the brain, heart,
adrenals, liver cells, and kidney tubules. Pulmonary hyperemia and
edema were also noticed. Animals killed 7-21 days post-application
of the compound showed evidence of the same type of degenerative
changes.
Monkeys dosed with various concentrations of hex in solution
exhibited discoloration of the skin which increased directly as the
concentration of hex applied increased. Swelling, oozing, and en-
crustation similar to that described above for rabbits were seen.
Healing eventually took place, but scarring and hair loss in the
area of application appeared to be permanent (Treon, et al. 1955).
Industrial Bio-Test Laboratories conducted a 28-day subacute
dermal toxicity study using albino rabbits. The study was reported
by Equitable Environmental Health (1976) but the data were not made
available for review.
Rats, rabbits, guinea pigs, and mice exposed to vapors of hex
showed signs of extreme irritation of the eyes and mucous membranes
(Treon, et al. 1955). At very high concentrations (46.5 ppm) ani-
mals responded by rubbing their noses with their forefeet, closing
C-23
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their eyes and retracting their heads. This behavior was accompa-
nied by sneezing, tearing, and irregular breathing. In less than
30-60 minutes the animals were gasping for breath.
Lower concentration of hex vapor (12.4 and 13.8 ppm) produced
similar irritation of the mucous membranes, although somewhat mild-
er in degree. The same symptoms were even seen at the low dosages
(1.0 and 1.6 ppm), but the symptoms developed over a period of
hours rather than minutes. Exposure to very low concentrations
(0.33 ppm and 0.15 ppm) resulted in some irritation of the eyelids
and increased respiratory rate. In the case of the latter dosage
(0.15 ppm), irritation was seen only in the mice, which developed
mild respiratory changes (Treon, et al. 1955). Rats which survived
the vapor exposure sessions lost weight and many of these animals
failed to regain their initial weights as long as six to eight
weeks after cessation of the exposures.
At autopsy Treon, et al. (1955) reported degenerative changes
similar to those described above (oral and dermal administration
experiments) in all species of animals tested. Prolonged intermit-
tent exposure to vapor concentrations as low as 0.15 ppm hex in-
duced slight degenerative changes in the livers and kidneys in all
species of animals employed.
Equitable Environmental Health (1976) also reported results
from two vapor toxicity studies, an acute test and a 28-day sub-
acute test which were conducted by Industrial Bio-Test Laboratories
(IBT) for Hooker Chemical and Plastics Corporation but public re-
view of the test data was not allowed.
C-24
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Synergism and/or Antagonism
There does not appear to be any information available on syn-
ergistic or antagonistic effects between hex and other compounds.
Teratogenicity
International Research and Development Corporation (IRDC,
1978) has recently completed a pilot teratology study using preg-
nant Charles River (CD) rats. Negative findings with respect to
teratogenic effects were reported for oral hex dosages up to 100
ing/kg/day.
The test protocol employed in the pilot teratology study in-
volved administration of various dosages of hex to 30 female
Charles River (CD) rats approximately 12 weeks of age. Females
were mated with male rats of the same strain. After mating, the
females were assigned to six groups, one control and five treatment
groups of five rats each. Hex was dissolved in corn oil and admin-
istered by gavage from day 6 through day 15 of gestation. Dosage
levels of 3, 10, 30, 100, and 300 mg/kg/day were administered to
the test groups and the control group was given the vehicle (corn
oil) on a comparable regimen of 10 ml/kg/day.
During gestation, the females were observed for clinical signs
of toxicity, mortality, and body weight gains. They were then sac-
rificed on gestation day 20 and the uterine contents examined for
viable and nonviable fetuses, early and late resorptions, and total
implantations. There were no differences in the four treatment
groups given 100 mg/kg/day or less when compared to the control
group in terms of number of viable or nonviable fetuses, resorp-
tions, implantations, or corpora lutea. Rats receiving doses of 3
or 10 mg/kg/day showed no treatment-related changes in appearance
C-25
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or behavior. Rats receiving 30 mg/kg/day or higher showed staining
of the anogenital area and reduced body weight gains. The females
in the 100 mg/kg/day group had body weight losses during the first
three days of treatment and reduced weight gains for the remainder
of the study. Survival was 100 percent for all rats given 100
mg/kg/day or less. All rats in the 300 mg/kg/day group were dead by
gestation day 10.
Various reproductive parameters examined in the pilot terato-
logy study are shown in Table 3.
Mutagenicity
Hex has been tested for mutagenicity and reported nonmutagenic
in both short-term ^n vitro mutagenic assays (National Cancer
Institute, 1977; Industrial Bio-Test Laboratories, 1977; Litton
Bionetics, 1978a) and in a mouse dominant lethal study (Litton
Bionetics, 1978b).
The National Cancer Institute (NCI, 1977) reported that pre-
liminary results indicated that hex was nonmutagenic in Escherichia
coli K12 (mutation site not specified) in the presence of a mam-
malian metabolic activation system containing mouse liver micro-
somes.
Negative results were also reported by Industrial Bio-Test
Laboratories (1977) using a test protocol almost identical to the
Ames mutagenic assay (Ames, et al. 1975). The tests used four
strains of Salmonella typhimurium with and without metabolic acti-
vation. Hex was dissolved in acetone and added to the microbial
assay plates in dosages from 10-5,000 ug/10 ul. Concentrations
greater than 10 ug/10 yl produced a bactericidal effect in three of
C-26
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TABLE 3
Pilot Teratology Study in Rats: Caesarean Section
Data For Individual Females*
o
I
Dosage Level
Control:
Total
Mean
3 mg/kg/day;
Total
Mean
10 mg/kg/day:
Total
Mean
30 mg/kg/day:
Total
Mean
100 mg/kg/day:
Total
Mean
300 mg/kg/day:
Dam Number
73758
77324
77333
77417
77445
Non-
Viable viable Late Re- Early Re-
Fetuses Fetuses sorptions sorptions
65
13.0
76
15.2
68
13.6
56
11.2
68
13.6
Died,
Died,
Died,
Died,
Died,
0
0.0
0
0.0
0
0.0
0
0.0
0
0.0
gestation
gestation
gestation
gestation
gestation
0 4
0.0 0.8
0 1
0.0 0.2
0 3
0.0 0.6
0 1
0.0 0.2
0 2
0.0 0.4
day 9 - gravid
day 10 - gravid
day 10 - gravid
day 10 - gravid
day 10 - gravid
Post Implan- Implan- Corpora
tation Loss tations Lutea
4 69 80
• w ^ \f \i
0.8 13.8 16.0
1 77 82
0.2 15.4 16.4
3 71 73
0.6 14.2 14.6
1 57 65
0.2 11.4 13.0
2 70 70
0.4 14.0 14.0
*International Research and Development Corp., 1978
-------
the four strains tested; a possible lethal effect occurred at 2,500
ug/10 ul or greater in the fourth strain. A repressive effect was
noted in three of the four strains at concentrations below 10 ug/10
ul. Volatilate (volatile vapors) of hex was also tested on one
strain using the vapor from hex concentrations of up to 2,500 yg/10
pi and exposure times of up to two hours. Results from two succes-
sive assays in the absence of rat liver enzymes (hex concentrations
10, 25, 50, 75, and 100 ug/10 uD were negative in all four tester
strains. Two assays using the same dosages in the presence of rat
liver microsomes were reported nonmutagenic; similarly, negative
results were obtained for the hex effusate as well. The investi-
gators expressed concern over the repressive effect of hex on the
test bacteria, stating "It appears that hex is probably nonmuta-
genic and that some toxic effect prevailed with respect to the
tester strains required for this assay. Analysis of variance and
multiple comparison of the data confirms this observation."
Litton Bionetics (1978a) conducted a mouse lymphoma cell assay
in order to evaluate the capability of hex in inducing specific
locus forward mutation. The indicator cells used in the assay were
Fischer mouse lymphoma cells derived from cell line L5178Y. These
cells are heterozygous for a specific autosomal mutation at the TK
locus and are bromodeoxyuridine (BUdR) sensitive. Scoring for
mutation is based on selecting cells which have undergone forward
mutation from a TK+/- to a TK-/- genotype by cloning them in soft
agar with BUdR. Cells were maintained in Fischer's medium for leu-
kemic mouse cells with 10 percent horse serum and sodium pyruvate.
The dosages used in the test were predetermined by exposing the
C-28
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cells to a wide range of hex concentrations and measuring the re-
duction of growth potential following a 4-hour exposure at each
dose. The maximum dose selected was that which produced a 50 per-
cent reduction in growth. The actual hex dosages employed were:
0.00040 ill/ml;
0.00048 ul/ml;
0.00056 ul/ml;
0.00064 ul/ml; and
0.00125 ul/ml
in the activated series (mouse liver microsomes were added to the
growth medium). A nonactivated series using somewhat lower dosages
was included also.
Both negative and positive controls were used; the negative
control for both series was the solvent dimethylsulfoxide (DMSO),
whereas ethyl methanesulfonate (EMS) and dimethylnitrosamine (EMN)
were used as positive controls in the nonactivated and activated
systems, respectively. Hex was added to the cells in the growth
medium for four hours. The cells were then washed and allowed to
express in the growth medium for three days. After the expression
period, results were evaluated by counting the TK-/- mutants after
cloning the cells in a selection medium (soft agar with BUdR).
Hex dissolved in DMSO was evaluated over the concentration
range of 0.0000025 ul/ml to 0.00125 ul/ml. Considerable toxicity
occurred at concentrations greater than this and the extent varied
according to the presence of the mouse liver activation system as
shown in Table 4. No cells treated with hex (at the concentrations
shown) survived in the non-activated system.
C-29
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TABLE 4
Summary of Mouse Lymphoma (L5178x) Results*
O
I
co
o
A. Name or code designation of the test compound: Hexachloropentadiene
B. Solvent: DMSO
NOTE: Concentrations are given in microliters (ul) or micrograms (ug) or nanoliters (nl) per milliliter.
TEST
NONACTIVATION
Solvent control
Negative control
EMS . 5ul/ml
ACTIVATION
Solvent control
Negative control
DMN . 5ul/ral
Test compound
0.00002 ul/ml
0.00004 ul/ml
0.00008 ul/ml
0.00016 ul/ml
0.00032 ul/ml
S-9
Source Tissue
mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
liver
liver
liver
liver
liver
liver
liver
liver
Relative
Suspension
Daily Counts Growth (%
(Cells/ml x 10ES) of control)
1
16.8
13.2
9.0
15.2
14.2
7.2
16.8
13.0
12.4
13.6
18.2
2
10.2
12.0
9.2
9.6
13.0
7.6
9.0
12.4
9.8
13.8
9.0
3
13.8
15.0
11.8
13.2
10.6
8.2
10.6
9.6
16.2
7.4
10.0
100.0
100.5
41.3
100.0
101.6
23.3
83.2
80.3
102.2
72.1
85.0
Total
Mutant
Clones
48.0
48.0
597.0
55.0
39.0
322.0
99.0
50.0
55.0
45.0
38.0
Relative
Total Cloning
Viable Efficiency
Clones (% of control)
257.0
234.6
89.0
281.0
293.0
55.0
288.0
269.0
194.0
359.0
309.0
100.0
91.1
34.6
100.0
104.3
19.6
102.5
95.7
69.0
127.8
110.0
Percent K • Mutant
Relative Frequency***
Growth** (X 10E-6)
100.0
91.5
14.3
100.0
105.9
4.6
85.3
76.9
70.6
92.1
93.5
18.7
20.5
670.8
19.6
13.3
585.5
34.4
18.6
28.4
12.5
12.3
* Source: Litton Bionetics, 1978a
** (Relative suspension growth X relative cloning efficiency) / 100
***(Mutant clones/viable clones) X 10E-6
-------
Hexachlorocyclopentadiene did not induce forward mutation in
L5178Y cells. The data presented in Table 4 show the concentra-
tions of the test compound employed, the number of mutant clones
obtained, surviving populations after the expression period, and
calculated mutation frequencies. No dose-related trends in either
absolute number of mutants or mutant frequencies were observed, and
at no level did any of the test parameters increase significantly
over the spontaneous level. Consequently, hex was considered to be
nonmutagenic under the conditions of this assay.
The mutagenic properties of hex were also evaluated in a domi-
nant lethal study of mice (Litton Bionetics, 1978b). The dominant
lethal assay provides a means of determining whether a compound is
capable of inducing damage in the germ cells of treated male mice.
Dominant lethality is manifested in various forms of fetal wastage,
both pre-and post-implantation. Positive dominant lethal assays
indicate that a compound is able to reach the developing germ
cells. Chromosome aberrations including breaks, rearrangements,
and deletions as well as ploidy changes and nondisjunction are be-
lieved to produce positive results on this test. Since substances
capable of producing gross chromosomal lesions are probably capable
of producing more subtle balanced lesions or specific locus muta-
tions, the test also provides suggestive evidence of nonlethal
mutations transmissible to future generations as well.
Litton Bionetics reported negative results, that is, there was
no evidence of significant dominant lethal activity by hex in mice.
The test protocol called for the assignment of ten random bred male
mice to one of five groups. Three test groups received hex at dos-
C-31
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ages of 1.0 mg/kg, 0.3 mg/kg, and 0.1 mg/kg, respectively. These
dosages were determined by deriving an LD5Q level (1.0 mg/kg) and
taking one-third and one-tenth of that dose. A fourth group re-
ceived only the solvent and the fifth group served as a positive
control. Hex was administered to the three experimental groups and
to the solvent control group by gavage for five consecutive days.
The positive control group received a known mutagen, triethylene-
melamine (TEM) in a single intraperitoneal injection. Two days
following treatment, each male was caged with two unexposed virgin
females. At the end of seven days, these females were removed and
replaced by two unexposed virgin females. This mating cycle was
continued for seven weeks. Each pair of female mice was killed two
weeks after mating and necropsied. Their uterine contents were
examined for dead and living fetuses, resorption sites, and total
implantations. All test parameters [fertility index, average im-
plantations per pregnancy, average resorptions (dead implants) per
pregnancy, proportion of females with one or more dead implanta-
tions, proportion of females with two or more dead implantations,
and the ratio of dead implantations to total implantations] were
within normal limits based on historical and concurrent control
levels for this test. Thus, there was no evidence of dominant
lethal activity in any of the hex treated groups. The positive
control group, however, did show the expected dominant lethal
activity.
Carcinogenicity
Various types of evidence may be used in evaluating the pos-
sible carcinogenic activity of a substance. In order of prefer-
C-32
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ence, these include: (1) human data; (2) animal data; (3) short-
term (in vitro) tests; (4) metabolic pattern; and (5) structure-
activity relationships. This section summarizes what is known
about each of the above.
No epidemiologic studies or case reports examining the rela-
tionships between exposure to hex and cancer incidences could be
found in the literature. As indicated previously, Hooker Chemicals
and Plastics Corporation reports that an in-house study of the mor-
tality patterns of hex-exposed workers is now underway; however,
the study is far from being completed (Zavon, 1978, personal commu-
nication) . Other in-house studies of workers employed in the manu-
facture of pesticides (including hex) are reportedly being conduct-
ed by Velsicol Chemical Corporation. We were unable to obtain any
further information on the current status or findings of these
studies.
The National Cancer Institute concluded that toxicologic
studies of hex in animals have not been adequate for evaluation of
carcinogenicity (NCI, 1977). Chronic toxicity studies as reported
in the Toxic Symptoms and Pathologic Effects section, were based on
too few animals in some cases and/or the duration of the experi-
ments was too short for appropriate evaluation of chronic effects,
including carcinogenicity.
Only one short term iri vitro test of hex for carcinogenic
activity could be identified.
Litton Bionetics (1977) reported the results of a test to
determine whether hex could induce malignant transformation in
BALB/3T3 cells _in vitro. The cells and methodology of the test
C-33
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were those of Dr. T. Kakunaga (1973), described elsewhere. The
basic rationale of the test and its validity as an indicator of
carcinogenic activity were described by the investigators as
follows:
The endpoint of carcinogenic activity is determined by
the presence of fibroblastic-like colonies which are
altered morphologically in comparison to the cells ob-
served in normal cultures. These (transformed) cells
grow in criss-cross, randomly oriented fashion with over-
lapping at the periphery of the colony. The colony exhi-
bits dense piling up of cells. On staining, the foci are
deeply stained and the cells are basophilic in character
and variable in size. These changes are not observed in
normal cultures, which stain uniformly.
Cell cultures with very little or no spontaneous transforma-
tion are maintained for use in these tests. The data generated at
each dose level of the test material are analyzed using the t sta-
tistic. A significant set of data for any dose level may be suffi-
cient to indicate a positive response. Because this assay is still
nonroutine, and definitive criteria for evaluation have yet to be
developed, scientific judgment and expert consultation are needed
for appropriate interpretation of results.
The BALB/3T3 cells used in the test were grown in Eagle's
minimal essential medium (EMEM) supplemented by 10 percent fetal
calf serum. Cultures were passaged weekly in 60 mm culture dishes.
Approximately 10,000 cells were seeded into 50 ml sterile tissue
culture flasks and incubated in EMEM to permit attachment. After
the cells were attached, the control and test compounds were added
to the plates. Dosages of 0.00001 ul/ml; 0.00002 ul/ml; 0.000039
Ul/ml; 0.000078 ul/ml; and- 0.000156 yl/ml of hex were employed.
The maximal dosage, 0.000156 ul/ml, was determined by selecting
C-34
-------
from preliminary cytotoxicity tests the maximum dosage which per-
mitted survival of at least 80 percent of the cells. 3-Methyl-
cholanthrene at 5 yg/ml was used as a positive control and the test
compound solvent was used as a negative control. Ten replicates
per dose level were prepared and chemical exposure was maintained
for 48 hours. Plates were then washed free of the compound and re-
plenished with fresh growth medium. The plates were then incubated
for an additional three to four weeks with twice weekly medium
changes. Cell integrity was monitored by daily observations.
Cells were separated from the medium, washed with saline, and
stained. They were examined for stained foci; all potential foci
were examined microscopically. Results were presented as the num-
ber of foci per set of replicate plates at each dosage level.
The test material was quite toxic to cells as indicated in the
preliminary range-finding tests. No significant carcinogenic
activity for hex was reported under the conditions of this test. A
low level of spontaneous transformation was observed on all of the
plates. Only the 3-methylcholanthrene treated plates showed a sig-
nificantly higher number of transformed foci than the negative
control.
It should be noted that in this and other cell culture tests,
extremely low dosages of hex were used. Because hex is relatively
toxic to cells in culture and test protocols normally require a
high survival rate, the applicability of test results to environ-
mental conditions is unclear. Taken together, however, the muta-
genicity and carcinogenicity tests conducted by Litton (1977,
1978a) suggest that toxicity, rather than chronic effects, is per-
C-35
-------
haps the critical effect of hex, even at very low dosages. Ex-
tremely poor survival has also been problematic in several sub-
chronic tests of hex in mammalian species.
A very recent study involving chronic dietary exposure of rats
to hexachlorobutadiene also provides some insight into the rela-
tionship between direct toxic effects and chronic effects (i.e.,
carcinogenesis) in this related compound (Kociba, et al. 1977).
Male and female Sprague-Dawley rats were maintained on diets
supplying 20, 2.0, 0.2, or 0 mg/kg/day of hexachlorobutadiene
(HCBD) for up to two years. Rats ingesting 0.2 mg/kg/day had no
discernible ill effects that could be attributed to this dose
level. Ingestion of the intermediate dose level of 2.0 mg/kg/day
caused some degree of toxicity, affecting primarily the kidney in
which increased renal tubular epithelial hyperplasia was noted.
Urinary excretion of coproporphyrin was also increased at this dose
level. Ingestion of the highest dose level of 20 mg/kg/day caused
a greater degree of toxicity. Effects included decreased body
weight gain and length of survival, increased urinary excretion of
coproporphyrin, increased weights of kidneys, and renal tubular
adenomas and adenocarcinomas, some of which metastasized to the
lung. In this study irreversible toxicological effects, such as
the development of neoplasms, occurred only at a dose level which
caused significant tissue injury and other manifestations of toxi-
city. No neoplasms resulted with dose levels which caused no in-
jury or only mild, reversible injury.
Little information is available on the metabolism of hex. Al-
though at least four metabolites were found in the Mehendale (1977)
C-36
-------
study, at the time of this writing they had not been identified.
Thus, the metabolic pathway is uncertain.
As far as structure/activity relationships are concerned, the
National Cancer Institute (1977) speculated that as a cyclopentene
vinyl halide, hex potentially may be metabolized to an electro-
phile. In addition, hex is related to the pesticides dieldrin,
heptachlor, and chlordane which have been found to induce liver
tumors in mice following oral administration (NCI, 1977).
Hex has recently been selected for testing in the National
Cancer Institute's test program (NCI, 1977). The reasons given for
its selection include: (1) its high potential for exposure (as an
industrial intermediate used in the manufacture of pesticides,
flame retardants and dyes, Pharmaceuticals, resins, and' germi-
cides) ; (2) its suspect chemical structure; and (3) the relative
lack of information on the effects of chronic exposure to this com-
pound.
Extremely limited data are available concerning the effects of
hex exposure on humans. That which is known about acute human
toxicity is based largely upon isolated spills or other accidental
incidents involving pesticide workers, laboratory technicians, or
others having occupational contact with hex. A recent incident in
which approximately 200 sewage treatment plant workers were exposed
to acutely toxic levels of hex from the illegal disposal of large
quantities of the compound has done much to elucidate the potential
health effects of acute human exposures. Due to the accidental and
episodic nature of these incidents and the lag time in setting up
environmental monitoring equipment in response to the incidents, it
C-37
-------
has not been possible to measure environmental concentrations of
hex at the exact time workers report symptoms (post-exposure sampl-
ing results are sometimes available). Thus, while there is infor-
mation regarding the range and variety of toxic responses, the
exact dose which elicited a given response remains unknown. It is
obvious that reliable dose-response estimates require accurate mea-
surement of both dose and response parameters.
Likewise, virtually nothing is known regarding the potential
effects resulting from chronic exposure to environmental sources of
hex. Potential modes of environmental exposure (e.g., through ex-
posure to contaminated air or water) are uncertain at this time.
According to Hooker's material safety data sheet for hexa-
chlorocyclopentadiene (Hooker Ind. Chem. Div., 1972), the compound
is very irritating to the eyes and mucous membranes causing lacri-
mation, sneezing, and salivation. Repeated contact with the skin
can cause blistering burns, and inhalation can cause pulmonary
edema. Hex is readily absorbed through the skin. Ingestion can
cause nausea, vomiting, diarrhea, lethargy, and retarded respira-
tion. Recommendations for safe use include: (1) good general
ventiliation plus local exhaust at points of potential fume emis-
sion; (2) respiratory protection of the organic vapor-acid gas
canister type and full-face self-contained breathing apparatus for
emergencies; (3) elbow-length neoprene gloves; (4) eye protection
including chemical safety glasses, plus face shield where appro-
priate; and (5) protective clothing including full-length clothing
fastened at neck and wrist, rubber safety shoes or boots, rubber or
other impervious clothing or aprons as needed for splash pro-
tection.
C-38
-------
According to Treon et al. (1955) , a very faint odor of hex was
detectable in air by some individuals at concentrations as low as
0.15 ppm which was the lowest concentration employed in their ex-
periments. At approximately twice that concentration (0.33 ppm), a
very pronounced, pungent odor was present.
Treon, et al. (1955) observed that headaches developed among
laboratory workers following incidental exposure to hex vapor from
the respiratory chambers used for their vapor inhalation experi-
ments. The exact concentration of hex escaping into the laboratory
from the opening of the respiratory chamber is unknown; however,
the chamber was not opened until the contaminated air had been ex-
hausted and the chamber flushed for some time with clean air.
Thus, the ambient concentration producing headaches among the labo-
ratory workers was well below the dosages employed in the animal
experiments. Because no mention is made of any other irritative
symptoms (e.g., lacrimation, etc.), it seems reasonable to specu-
late that the concentration of hex present was somewhere in the
range between 0.15 ppm-1.0 ppm, above the detection threshold but
below the level producing acute symptoms of irritation.
Irritant effects are elicited at a vapor concentration greater
than that shown to produce chronic toxicity in animals. Thus,
Treon et al. (1955) concluded that the irritant effects of hex
vapors are not sufficiently pronounced to serve as a warning that a
hazardous level of hex vapor is present and/or that hazardous expo-
sure is taking place.
According to Naishstein and Lisovskaya (1965), hex may be de-
tected by taste and smell at very low concentrations in water.
C-39
-------
They placed the threshold level for altering the organoleptic qual-
ities of water at 0.0014-0.0010 mg/1.
Epidemiologic Studies
-*•
To date, the only well documented incident of the acute toxi-
city of hex to humans occurred at the Morris Forman Wastewater
Treatment Plant (MFWTP) in Louisville, Kentucky. The problem ap-
parently began about the middle of March, 1977, when an unknown
chemical, later identified as a mixture of hex and octachlorocyclo-
pentene (Table 5), began entering the Morris Forman sewage treat-
ment facility. An exact date of initial appearance at the plant,
and hence, the initial date of worker exposure is unknown. How-
ever, unusual odors became evident around March 17, 1977.
The odor gradually intensified over the next two weeks. From
March 25-28, an odoriferous, sticky material entered the plant and
gummed the barscreens and grit collection systems in the primary
treatment area. Attempts to dislodge the material with steam pro-
duced a blue gas which permeated the grit removal and sludge handl-
ing areas. Workers exposed to this vapor complained of severe
irritation of the eyes, nose, throat, and lungs (Carter, 1977b).
Approximately 20 workers sought medical treatment for tracheobron-
chial irritation. These workers were treated in the local emer-
gency room; none were hospitalized (Singal, 1978).
A sample of the material from the Screen and Grit Building was
sent to the U.S. EPA Laboratory in Athens, Georgia, for analysis.
The primary contaminants in the samples were identified as hexa-
chlorocyclopentadiene (hex) and octachlorocyclopentene (octa).
Octa is a waste by-product in the manufacture of hex whose toxicity
C-40
-------
TABLE 5
Analysis of a Sludge Sample Obtained in the
Screen and Grit Building on April 2, 1977,
Morris Forman Wastewater Treatment Plant,
Louisville, Kentucky*
Compound"
Concentration - % by weight
Octachlorocyclopentene
Hexachlorocyclopentadiene
Hexachlorobenzene
Pentachlorobenzene
Octachloronaphthalene
Heptachloronaphthalene
Hexachloronaphthalene
Mirex
9
4
0.3
0.2
0.4 (estimated)
0.2 (estimated)
(not quantitated)
0.007 (estimated)
*Source: Singal, 1978
•'•Analysis was conducted by the U.S. Pood and Drug Administration,
Division of Chemical Technology, Chemical Industry Practices
Branch
2
The sample was analyzed using gas chromatography interfaced with
mass spectroscopy for positive identification of each compound
C-41
-------
is presently unknown. Table 5 shows the results of the analysis.
Due to the apparent potential toxicity of hex (and the unknown
toxic potential of octa), the sewage treatment plant was evacuated
and closed on March 29, 1977. Thereafter, until the partial re-
opening in June, 1977, 105 million gallons per day of domestic and
industrial wastes were diverted directly to the Ohio River.
Estimates of the extent of contamination indicate that about
60 million gallons (25,000 tons) of hex-contaminated material were
present at the Morris Forman plant. Of this, approximately 6 tons
of hex and octa were thought to be present in the contaminated
waste. U.S. EPA's analysis revealed hex concentrations up to 1,000
ppm in the sewage water at the time of the plant closure. The route
of chemical contamination was traced to one large sewer line which
passed through several heavily populated areas. Wastewater in this
sewer showed hex and octa in concentrations ranging up to 100 ppm.
Samples from the sewer showed air concentrations ranging up to 0.4
mg/1 for hex and up to 0.03 mg/1 of octa. Thus, it was decided to
investigate the health of not only the workers at the sewage treat-
ment plant but also residents of the area surrounding the sewer
line (Morse, et al. 1978).
A cooperative investigation involving Region IV, U.S. EPA
(Surveillance and Analysis Division), Center for Disease Control
(CDC), National Institute for Occupational Safety and Health
(NIOSH), Jefferson County (Kentucky) Health Department, and the
Kentucky State Health Department was initiated.
Information on both aspects of the investigation (i.e., com-
munity residents on one hand and exposed workers on the other) is
C-42
-------
thus far unpublished but preliminary drafts of reports were made
available by Dale Morse, M.D., who headed the initial epidemiologic
studies conducted bj the Center for Disease Control (Morse, et al.
1978) and by Mitchell Singal of the Hazard Evaluation and Technical
Assistance Branch of NIOSH who reported on the follow-up investiga-
tions of workers during cleanup operations at the sewage treatment
facility (Singal, 1978). Findings from these drafts are reported
below; however, they should be regarded as preliminary.
The Center for Disease Control investigation began by identi-
fying all sewage treatment employees who worked at the plant for
two or more days during the period from March 14-29, 1977. Health
effects evaluations, including mailed questionnaires, physical
examination, and blood and urine testing, were conducted appropri-
ately to exposed individuals who agreed to participate. The ques-
tionnaire covered demographic information, a detailed work-area
history, symptoms and history of chemical poisoning, personal
habits, and other sources of chemical exposure. Routine tests were
performed on blood and urine specimens. Additional samples were
sent to NIOSH laboratories for potential toxic chemical analysis.
Of 193 plant employees who had worked during the latter half
of March, questionnaire data were obtained from 145. Seventy-five
percent of the questionnaire respondents indicated that they
detected an unusual odor at the plant sometime during March. A few
individuals reported detecting unusual odors as early as March 1,
1977; the percentage reporting the odor by March 14 was noticeably
C-43
-------
increased. From March 15 onward, the percentage of workers who re-
ported noticing the odor steadily increased until the plant was
closed on March 29.
A comparison between the time of odor detection and the onset
of eye irritation, the most common symptom, showed that irritation
developed on the same day in 45 percent of individuals, within 1 to
5 days in 28 percent, and after five days in 21 percent. Only 6
percent of employees reported onset of symptoms prior to noticing
an unusual odor at the plant.
Eye irritation, headache, and throat irritation were the most
common symptoms, with 59 percent, 45 percent, and 27 percent of
employees reporting these symptoms, respectively. Data for these
and other symptoms are reported in Table 6. Of 41 workers physi-
cally examined, five had signs of eye irritation (tearing and/or
redness) and five had signs of skin irritation.
Forty-two persons were interviewed and provided blood and
urine samples. This included 24 of 29 (83 percent) of the workers
who had been previously evaluated by local physicians, 17 of 164
other plant employees (a 10 percent random sample) as well as one
non-employee accidentally exposed to the contaminated sludge.
Abnormalities were found in laboratory analysis of some of the
workers (e.g., LDH elevations in 27 percent and proteinuria in 15
percent of those examined). No LDH or urinalysis abnormalities
were corroborated on repeat tests run three weeks later by another
laboratory. Also, no abnormalities were reported among individuals
seen at the local hospital or by the plant physician.
C-44
-------
o
I
*»
en
TABLE 6
Symptoms of 145 Plant Employees,
Louisville, Kentucky, March, 1977*
Symptom
Eye irritation
Headache
Throat irritation
Nausea
Skin irritation
Cough
Chest pain
Difficult breathing
Nervousness
Abdominal cramps
Decreased appetite
Decreased memory
Increased saliva
Number
with Symptom
86
65
39
31
29
28
28
23
21
17
13
6
6
Percent
with Symptom^,
59
45
27
21
20
19
19
-L «/
16
14
12
9
4
4
*Source: Morse, et al. 1978
-------
Detailed work area histories on 124 individuals during the
highest exposure period showed that "cases" occurred in all areas
of the plant. A cas£ was defined as an individual who reported two
or more major symptoms (eye irritation and headaches) or one major
symptom and two minor ones (sore throat, cough, chest pain, diffi-
culty breathing, skin irritation). Attack rates were significantly
higher for individuals who had been exposed to the screen and grit
chamber (p .0001) and to the primary settling area (p .02) than
for workers not exposed to these areas.
Analysis of data according to employee work areas revealed
that symptoms occurred in workers of all job categories and in all
work areas. Data for attack rates in employees by main work area
are reported in Table 7. Only small differences in case rates
appeared by work area although the highest attack rates occurred in
workers in the primary treatment area where the level of hex was
2
presumably highest. Attack rates were significantly higher by X
(chi-square) test for individuals who had been exposed to the
screen and grit chamber (p » .0001) and to the primary settling
area (p = .02) than for workers not exposed to these areas.
The initial investigation demonstrated that 64 of 145 (44 per-
cent) of current employees questioned at the wastewater treatment
plant had experienced headache and mucous membrane, skin, and
respiratory tract irritation after exposure to airborne hex. High-
est attack rates occurred among workers in the primary treatment
area where exposure was highest and ventilation poorest. In most
cases symptoms were transient, but in some workers, they persisted
for several days. This episode clearly demonstrates the volatility
C-46
-------
TABLE 7
Attack Rates in Employees by Main Work Area,
Louisville, Kentucky, March, 1977*
o
i
Main Work Area
Primary treatment
Throughout plant
Vacuum filtration
Secondary aeration
chamber
Administration and
laboratory
Final effluent
pump station
Low pressure
oxidation
Incineration
Totals
Number of
Employees
19
71
19
14
30
10
13
17
193
Number
Reporting
Symptoms
17
54
15
12
22
5
10
10
145
Percentage of
Employees Re-
porting Symptoms
89
76
79
86
\J \j
73
50
77
/ /
5£
75
Percentage of Cases
of Those Reporting
Symptoms
IK'Q
'Sy
AO
1O
47
1 1
A ">
4z
41
*s X
40
in
JU
20
44
*Source: Morse, et al. 1978
-------
of hex and its potential for having a toxic effect on humans. Re-
sults of the follow-up investigation of the sewage treatment plant
workers and the community survey are reported below.
After the initial health evaluation survey was completed
(April 3, 1977), NIOSH assumed the responsibility for follow-up
investigations of the sewage treatment workers exposed during the
March, 1977, episode. NIOSH was also responsible for medical moni-
toring of those involved in the cleanup operations prior to reopen-
ing the Morris Forman plant. NIOSH1s activities consisted of the
following: (1) administering follow-up questionnaires to all plant
employees to determine how persistent symptoms had been after the
initial chemical exposure in March; (2) review of the medical
records of the 90 employees who had seen the plant physician from
late March through May 10, 1977; (3) collection of repeat biologic
samples on the 23 employees who had shown some abnormality on the
testing done by the CDC physicians (March 31-April 2, 1977, tests);
(4) biological monitoring of EPA and NIOSH industrial hygienists
and environmental technicians exposed to the chemicals in the sewer
system during cleanup; and (5) medical monitoring of Morris Forman
plant employees who were actively involved in the plant cleanup.
Results of each of these aspects of the investigation are reported
below.
Usable responses were obtained from 182 individuals on the
follow-up questionnaire. The frequency of symptoms among those who
completed the questionnaire is shown in Table 8. In decreasing
order of frequency, these symptoms included eye irritation, head-
ache, fatigue, chest discomfort, sore throat, cough, nausea, and
skin rash. These symptoms were surprisingly persistent. Except
C-48
-------
TABLE 8
Symptoms Reported on Follow-up Questionnaire,* Morris Forman
Wastewater Treatment Plant, Louisville, Kentucky**
n
i
vo
Symptom
Headache
Eye Irritation
Sore Throat
Cough
Chest Discomfort
Skin Irritation
Nausea
Fatigue
% with Symptoms
in Last 2 Weeks
of March***
55%
62%
30%
24%
34%
21%
22%
34%
Persistence of Symptoms***
Gone Within
1 Day
19%
36%
15%
14%
11%
18%
18%
8%
(% of those
Gone Within Gone Within
1 Week 2 Weeks
30%
23%
49%
27%
20%
18%
23%
16%
18%
16%
13%
16%
21%
10%
18%
24%
with symptoms)
Still Present
at Time
of Survey
32%
15%
18%
36%
39%
46%
25%
45%
* Distributed and collected last 2 weeks of May 1977
Excludes employees actively involved in cleanup, since their symptoms could relate to exposure
during cleanup instead of to exposure prior to the plant shutdown.
** Source: Singal, 1978
***Percentages do not quite add to 100% due to some employee confusion about the need to fill in
questionnaire completely.
-------
for eye irritation and sore throat, 25-45 percent of those who ex-
hibited symptoms during the last two weeks of March, 1977, still
had them six weeks later. Although symptoms occurred in workers in
all areas of the plant, maintenance department personnel consis-
tently reported the highest number of symptoms.
A review of medical records of the 90 workers examined by the
plant physician (mid-March to May 10, 1977) revealed symptom re-
ports similar to those reported on the NIOSH and CDC question-
naires. Fatigue, headache, and mucous membrane irritation were the
predominant complaints; respiratory and skin problems were also re-
ported. Seven of the 90 workers reported transient memory loss
ranging from a few minutes to a few days. These are believed to
represent a transient state of confusion, rather than true amnesia
(Singal, 1978). Although several workers reported neurologic symp-
toms, the plant physician found no one with any objective neuro-
logic signs. Seven persons had rash on exposed areas of face and
arms. Respiratory tract symptoms, cough, and chest discomfort were
commonly reported. Twenty-eight persons, including those with
respiratory symptoms, received chest x-rays. Essentially all of
the x-rays were normal. Sixteen persons received blood gas deter-
minations, none of which showed an elevated pCO^ or a pC^ below 70
mm Hg. Pulmonary function tests were done on 22 individuals but no
significant pattern of abnormalities was seen. Cholinesterase
levels on 27 workers were normal. Several workers had elevated
liver function tests; these were mainly minor elevations of lactic
dehydrogenase (LDH) and alkaline phosphatase which are difficult to
interpret. More specific liver function tests such as serum gluta-
C-50
-------
mic oxalacetic transminase (SCOT) and serum glutamic pyruvic trans-
aminase (SGPT) were elevated in three persons. Six elevations of
bilirubin, two elevations of serum creatinine, and six mild pro-
*
teinurias were detected. Unfortunately, the specimens were ana-
lyzed by at least three different laboratories and comparison/
interpretation of these results is uncertain. Attempts to develop
a technique to isolate and identify concentrations of hex in speci-
mens of blood or urine at the time of the investigation were unsuc-
cessful (Morse, et al. 1978).
Biological monitoring of NIOSH and U.S. EPA personnel who were
actively involved in the cleanup effort showed no significant
abnormalities.
Repeat laboratory tests were done on 20 of the 23 sewage
treatment plant workers who had abnormalities on the blood and/or
urine tests at the time of plant shutdown. Three of these people
continued to have persistent abnormalities in liver function tests
on one or more occasions but there were no persistent urinary ab-
normalities.
Exposure levels of the cleanup crew were monitored by taking
samples of breathing zone concentrations (inside masks) of hex and
octa. These values are reported in Tables 9 and 10.
Biological monitoring of the cleanup crew was also conducted
by NIOSH. Due to continuous turnover of crew members, it was not
possible to obtain pre-exposure baseline studies on more than 54
percent of the workers. Symptoms reported by crew members were
similar to those reported on the NIOSH and CDC questionnaire sur-
veys of the plant employees in March. Headache and eye irritation
C-51
-------
TABLE 9
'Personal Breathing Zone Concentrations of Hexachlorocyclopentadiene (HCCPD) and
Octachlorocyclopentene (OCCP) Measured in the Grit Loading and Screen and Grit Buildings
During Grit Removal, Morris Forman Hastewater Treatment Plant, Louisville, Kentucky, 1977**
O
I
en
to
Sample
Date
4-17
4-18
M
4-19
N
M
N
4-20
H
M
tl
M
N
Sample
No.
CR-022
CR-023
CR-024
CR-027
CR-029
CR-035
CR-036
CR-037
CR-038
CR-041
CR-042
CR-048
CR-049
Sample Description
PBZ-GLB:
PBZ-SGB:
PBZ-SGB:
PBZ-GLB:
PBZ-SGB:
PBZ-SGB:
PBZ-GLB:
PBZ-SGB:
PBZ-GLB:
PBZ-SGB:
PBZ-GLB:
PBZ-SGB:
PBZ-GLB:
Maintenance Mechanic
Equipment Operator
Equipment Operator
Loading Operator
Equipment Operator
Equipment Operator
Loading Operator
Equipment Operator
Loading Operator
Equipment Operator
Loading Operator
Equipment Operator
Loading Operator
Sampling Period
1257
1459
1528
2044
1536
2043
0805
1008
1243
0806
1007
1255
1645
1646
2045
0005
0230
0600
0006
0235
0600
0747
0950
1303
0749
0948
1317
1646
1858
- 1322
- 1533
- 1919
- 2222
- 1907
- 2220
- 0905
- 1120
- 1519
- 0909
- 1120
- 1515
- 1803
- 1805
- 2253
- 0119
- 0440
- 0740
- 0120
- 0441
- 0741
- 0857
- 1112
- 1359
- 0851
- 1113
- 1405
- 1715
- 2234
Sample Volume
liters
6
12
16
13
10
4
10
12
14
14
8
12
7
Environmental Criteria
' . . . ., . — - . . • • — = i 1 1 -— • — i ' • s ' — v—. 1 -• i i • — i y"i" •
Airborne
HCCPD
1.5
3
7
0.7
0.5
2.3
8
6
1
5
7
0.8
1.4
10
Concentration - ppb
OCCP
2.
1.
t
0.
1.
v
0.
3.
2
4
1.
1.
1.
1.
2.
4
2
• *
9
1
8
7
0
1
9
2
2
None Established
'Concentration was measured underneath the protective vinyl suft in tfi¥ breathing zone of the worker.
**Source: Singal, 1978
PBZ-GLB denotes personal breathing zone sample in the Grit Loading Building and PBZ-SGB denotes personal breathing zone
sample in the Screen and Grit Building.
Parts of contaminant per billion parts of contaminated air sampled by volume.
-------
TABLE 10
Personal Breathing Zone Concentrations of Hexachlorocyclopentadiene (HCCPD) and
Octachlorocyclopentene (OCCP) Measured Inside the Protective Suits Worn by Persons
Involved with the High Pressure Water Washdown of the Screen and Grit Building,
Morris Forman Wastewater Treatment Plant, Louisville, Kentucky, 1977*
Sample Date
4-22
4-22
4 -'2 2
9 4-23
en
w 4-23
4-23
4-25
4-25
4-25
Environmental
Sample No.
CR-058
CR-059
CR-060
CR-078
CR-079
CR-080
CR-081
CR-082
CR-083
Criteria
Sampling Period
0945
1225
0946
1225
0947
1226
0850
1248
0851
1253
1045
1252
1245
1438
1308
1438
1246
- 1117
- 1534
- 1122
- 1540
- 1120
- 1540
- 1145
- 1600
- 1145
- 1601
- 1145
- 1604
- 1401
- 1534
- 1405
- 1537
- 1402
Sample Volume
liters
12
15
13
18
9
8
32
23
29
Airborne
HCCPD
0.8
0.6
0.7
0.5
1.0
1.0
0.3
0.4
0.3
10
Concentrations - ppb
OCCP
4
* 0.9
1.1
0.8
1.8
1.4
0.4
0.6
0.5
None Established
*Source: Singal, 1978
Parts of contaminant per billion parts of contaminated air sampled by volume
-------
were the predominant symptoms; sore throat, fatigue, nausea, dizzi-
ness, chest discomfort, cough, and skin irritation were also re-
ported. Physical examinations on cleanup crew members were unre-
markable except for conjunctival irritation in workers wearing
half-face respirators.
Of 97 crew members tested, 18 (19 percent) showed some eleva-
tion of a liver function test results on one or more of the five
occasions testing was done (Singal, 1978). These elevations were
generally small (Table 11), but once they appeared, they tended to
persist over several weeks (Table 12). A small number of abnor-
malities appeared on renal function tests but generally these were
small and non-reproducible on serial testing. Likewise, abnormali-
ties in complete blood counts were also minor and non-reproducible.
It should be noted that the laboratory results on cleanup
workers are difficult to interpret due to lack of adequate con-
trols. Essentially all of the plant employees, including many of
the cleanup workers, had been exposed in March prior to the plant
shutdown. As indicated, there were no environmental samples taken
at the time of the acute exposure episode. Although exposure
levels of the cleanup workers were well below the current occupa-
tional standard for hex (0.01 ppm), one cannot rule out the possi-
bility that abnormalities among the cleanup crew are reflective of
earlier, unspecified exposures. Interpreting the significance of
variations in liver function tests of the magnitude seen in this
group of workers is difficult. First, many of the abnormalities
seen are relatively nonspecific, that is such changes may be caused
by a variety of conditions and thus are not necessarily
C-54
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TABLE 11
Abnormalities in Lab Tests on Cleanup Workers,
Morris Forman Wastewater Treatment Plant,
Louisville, Kentucky*
o
1
en
en
Lab Test
1 SCOT -
(serum glutamate-
oxalacetic acid
transaminase)
2 Serum alkaline
phosphatase
Ranges
of values
40-49
50-59
60-69
70-79
80-89
90-99
100-109
110-119
120-129
Number of Persons'
Results in Range
5
1
4
0
1
1
3
1
1
Normal Range
kr1
7-40 mU/ml
30-100 mU/ml
3 Serum total
Bilirubin
4 Serum LDH
(lactate
dehydrogenase)
5 Serum creatinine
1.0-1.9
230-239
1.3-1.9
0.15-1.0 mg%
100-225 mU/ml
0.5-1.3 mg/dl
*Source: Singal, 1978
-------
TABLE 12
Liver Function Abnormalities in Cleanup Workers, Morris Forman Wastewater
Treatment Plant, Louisville, Kentucky*
O
I
tn
Patient
No.
1
2
' 3
4
5
6
7
g
9
10
11
12
13
14
15
16
17
18
Date of Visit
4/8 4/12 4/20 5/5
SCOT
Dili
SCOT
Bill
SCOT 51 SCOT 63 SCOT
Alk phos
SCOT
Alk phos
SCOT 31 SCOT 31 SCOT
SCOT 43 SCOT 52 SCOT
Alk phos 105 Alk phos
SCOT 44
Alk phos 88 Alk phos 96 Alk phos
SCOT
Alk phos 103
LDH 232 LDH 159
SCOT 42
SCOT 59 SCOT 54 SCOT
46l
0.92
48
1.4
45
100
42
113
60
39
120
101
87
46
5/19
SCOT
Bill ,
Alk phos
SCOT
SCOT
LDH
SCOT
SCOT
SCOT
Alk phos
SCOT
SCOT
SCOT
Hours Spent
in Cleanup
35
1.6
117
47
66
239
43
63
39
129
93
47
48
40
56
115
150
11
100
5
80
110
40
80
60
80
15
32
108
40
140
Did Lab
Abnormality
Result in
Removal from
Cleanup?
, Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
No
No
Yes
Yes
No
Yes
No
Yes
*Source: Singal, 1978
1 SCOT = Serum glutamate-oxaloacetate transterase in mU/ml - Normal range = 7-40 mu/ml
2 Bili = Total serum bilirubin in mg% - Normal range = 0.15-1.0 mg%
3 Alk phos - Serum Alkaline phosphatase in mU/ml - Normal range = 30-100 mU/ml
4 LDH = Serum Lactate dehydrogenase in mU/ml • - Normal range = 100-224 mU/ml
-------
attributable to exposure. Second, there is little consensus concerning
what constitutes the normal range in some of these tests. Despite
these problems in analysis, Dr. Singal expressed the opinion that
these data suggest that exposure to the mixture of chemicals con-
taminating the sewage treatment plant may be associated with some
mild liver injury (Singal, 1978).
In a community survey, CDC workers administered a question-
naire to a systematically selected sample of residents in a
48-block area surrounding the contaminated sewer line (Morse, et
al. 1978). One home per block was surveyed by administering a
questionnaire to the head of each household. In all, 212 occupants
of the 48-block area were surveyed. Questions were asked concern-
ing basic demographic data, history of unusual odors, and any symp-
toms noted by household members within the past two weeks.
Results of the community survey were essentially negative.
Eight of the 212 persons (3.8 percent) reported noticing an unusual
odor at some time during the preceding two weeks. While some of the
respondents reported symptoms compatible with hex exposure (head-
ache, 4.7 percent; burning or watering eyes, 4.7 percent), no symp-
tom occurred at greater than background rates. Symptoms not asso-
ciated with hex were reported just as frequently as those possibly
related to exposure. Furthermore, there was no association between
symptom rates and distance from the sewer line. Subsequent air
sampling failed to show a significant ambient concentration of hex
in the sewer line area.
C-57
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CRITERION FORMULATION
Existing Guidelines and Standards
The Occupational Safety and Health Administration (OSHA) has
not set a standard for occupational exposure to hex. On the other
hand, the American Conference of Governmental Industrial Hygienists
(ACGIH) has adopted both a threshold limit value (TLV) and a Short
Term Exposure Limit (STEL) for hexachlorocyclopentadiene. The cur-
rent occupational TLV for hex is set at 0.01 ppm (0.11 mg/m3) ,
which, according to ACGIH "represents a time-weighted average con-
centration for a normal 8-hour workday or 40-hour workweek to which
nearly all workers may be repeatedly exposed, day after day, with-
out adverse effect" (ACGIH, 1977). The Short Term Exposure Limit
(STEL) for hex is set at 0.03 ppm (0.33 mg/m ). This level repre-
sents the maximal concentration to which workers can be exposed for
a period up to 15 minutes without suffering from irritation;
chronic or irreversible tissue damage; or narcosis of sufficient
degree to increase accident proneness, impair self-rescue, or
materially reduce work efficiency. The STEL should be considered a
maximum allowable concentration or absolute ceiling not to be ex-
ceeded at any time in the 15 minutes. Up to four excursions up to
the STEL are permitted per day provided that at least 60 minutes
occur between excursions up to the STEL (ACGIH, 1977).
In selecting the TLV and STEL values for hex, the ACGIH empha-
sizes that these particular levels were selected on the basis of
preventing irritant effects rather than chronic toxicity. The USSR
has recommended a tenfold lower limit (0.001 ppm) for occupational
exposures.
C-58
-------
No nonoccupational exposure limits have been established or
recommended except for one Soviet study which proposed a maximum
concentration of 0.001 mg/1 in water to prevent "organoleptic ef-
fects" (i.e., adverse effects on the taste and odor of water).
There is a serious lack of data to support nonoccupational exposure
limits or environmental criteria for hex. Specifically lacking
are: (1) epidemiologic studies of individuals having known and
quantifiable hex exposures; (2) long-term animal studies (e.g.,
2-year chronic feeding studies) suitable for evaluating chronic ef-
fects, especially carcinogenicity; (3) data on current levels of
human exposure from various media; and (4) suitable methods for
interpreting the significance of i.n vitro assays and their applica-
bility to actual environmental conditions. Without these essential
data it is not possible to use the model proposed by U.S. EPA's Car-
cinogen Assessment Group (GAG) to derive recommended exposure cri-
teria for humans. In fact, the GAG states that "there is insuffi-
cient evidence to categorize this compound as a carcinogen or non-
carcinogen." Consequently, other toxic endpoints must form the
basis for recommended exposure criteria until a more adequate in-
formation base on hex is developed.
Special Groups at Risk
As indicated earlier, it is presently unknown whether inges-
tion or inhalation of hex constitute significant sources of expo-
sure among the general population. Although it is not likely this
is the case, present data on the environmental occurrence of hex
are so sketchy that this possibility cannot be ruled out.
C-59
-------
Occupational exposures appear to constitute the only document-
ed source of human exposure to hex. Oral contact does not appear to
be a likely mode of occupational exposure. However, dermal and in-
halation exposures are recognized hazards for the following groups:
(1) workers engaged directly in hex manufacture; (2) those engaged
in the formulation and use of other, related pesticides where hex
may be present as an impurity; (3) workers dealing with flame re-
tardants; (4) those having "quasi-occupational" exposures such as
sewage treatment workers, industrial hygienists, etc.
Basis and Derivation of Criterion
Notwithstanding the obvious data deficiencies, some tentative
recommendations can be made in consideration of the levels of hex
which produce chronic toxicity in laboratory experiments.
As indicated earlier, there are no epidemiologic studies nor
suitable chronic toxicity studies in mammals from which threshold
levels for chronic effects could be derived. Very little is known
regarding potential hex exposures through ingestion of contaminated
food or water. In the environment hex has been detected only in
specific bodies of water near points of industrial discharges.
There are no data on hex levels in drinking or untreated water.
Based on the available and cited literature, there is insuffi-
cient evidence to categorize this compound as a carcinogen or non-
carcinogen. There has not been a satisfactory study of the effects
of chronic oral exposure to hex. A single study of chronic oral
toxicity has been reported by Naishstein and Lisovskaya (1965).
The test consisted of only one species (rats) and the duration of
exposure was only six months. No neoplasms were reported, however
C-60
-------
the duration of the study would not have been sufficient for a pro-
per evaluation of carcinogenicity.
Hex has been tested for mutagenicity and reported nonmutagenic
in both short-term iri vitro mutagenic assays (NCI, 1977; IBT, 1977;
Litton Bionetics, 1978a) and in a mouse dominant lethal study (Lit-
ton Bionetics, 1978b). No epidemiologic studies or case reports
examining the relationship between exposure to hex and cancer inci-
dences could be found in the literature. Therefore, there is
virtually no information regarding the carcinogenic potential of
chronic exposure to hex. In selecting hex for future chronic toxi-
city testing, National Cancer Institute (1977) recognized these
data voids.
Although one study (Treon, et al. 1955) reported on the ef-
fects of chronic low-dose inhalation of hex, its applicability in
deriving water quality guidelines is unclear. Furthermore, with
the exception of very limited data on hex in water near points of
discharge, there appears to be no information on hex levels in
water bodies. What is needed is a method for converting the re-
sults of respiratory exposure experiments into equivalent dosages
from water.
Stokinger and Woodward (1958) describe a model by which the
threshold limit values (TLVs) for industrial substances in air may
be used in establishing drinking water standards. The model as-
sumes that, for any given inhaled dose, an equivalent ingested dose
from ingested water can be derived using reasonable estimates of
daily air and water intakes and corresponding respiratory and gas-
trointestinal absorption rates. In the absence of suitable chronic
C-61
-------
ingestion studies of hex, a modified version of the Stokinger and
Woodward model (44 FR 15956) can be used to estimate suitable
limits for hex in water based on the established threshold limit
value expressed as milligrams per cubic meter of air.
The threshold limit of 0.11 mg/m3 (0.01 ppm) hex represents
what is believed to be a maximal concentration to which a worker
may be exposed for eight hours per day, five days per week over his
working lifetime without hazard to health or well-being (ACGIH,
1977). Three factors are applied to the TLV to arrive at an esti-
mate of allowable daily intake (ADI). The first factor is respira-
tory intake or respiratory volume during an 8-hour period (assumed
to be 7.6 m3, or one-third of the 24-hour respiratory volume of
23 m3) . The second term expresses the efficiency with which the
material is absorbed from the respiratory tract. In the case of
hex, as the absorption rate is unknown, 70 percent absorption is
assumed. The third term is a weighting factor for converting the
5-day per week occupational exposure (inherent in the TLV) to a
7-day per week equivalent in keeping with the more continuous pat-
tern of exposure to drinking water.
According to the model, the amount of hex that may be taken
into the bloodstream and presumed to be noninjurious and which,
hence, may be taken in water each day is:
ADI = (TLV) x (RI) x (RA) x (WF)
where:
TLV is the industrial Threshold Limit Value, 0.11 mg/m
(ACGIH, 1978)
RI is the r-espiratory intake term (respiratory volume
of 7.6 m pet 8 hour)
C-62
-------
RA is the respiratory absorption coefficient, here as-
sumed to be .70
WF is a weighting factor expressing the proportion of
the week exposed to the TLV, here assumed to be 5/7
or 0^7143.
(0.11) x (7.6) x (0.70) x (0.7143) = 0.4180 mg hex per day
Therefore, ADI = 0.4180 mg
If EPA's modified version of tne Stokinger and Woodward method is
used (44 FR 15956), we obtain:
CR = ADI / [WC + (R x F)]
where:
CR is the criterion for which we are solving
ADI is allowable daily intake derived from the TLV,
i.e., 0.4180 mg/day
WC is the volume of water ingested per day, i.e., 2.0
liters.
R is the bioconcentration factor for hex in fish, 4.34
F is the average weight of fish consumed per day,
0.0065 kg.
Solving for CR, we obtain:
CR = 0.4180 / [2.0 + (4.34 x 0.0065)]
CR = 0.2061 mg/1 or 206 yg/1
This value is included as an example of an acceptable limit, but it
is not being recommended as a criterion. According to Stokinger
and Woodward (1958), "This derived value represents an approximate
limiting concentration for a healthy adult population; it is only a
first approximation in the development of a tentative drinking
water criterion... Several adjustments in this value may be neces-
sary. Other factors, such as taste, odor, and color may outweigh
C-63
-------
health considerations because acceptable limits for these may be
below the estimated health limit."
It should also be noted that the basis for the above recom-
mended limit, the TLV for hex, is set on the basis of avoidance of
irritation, rather than chronic effects (ACGIH, 1977). Should
chronic effects data become available, both TLVs and recommenda-
tions based on them will warrant reconsideration.
A single study of chronic oral toxicity in white rats reported
no adverse effects (specifically changes in peripheral blood cells,
ascorbic acid content of the adrenals, conditioned reflexes of the
animals, or histological structure of the organs) following daily
oral administration of doses up to 0.2 ug/kg (4 yg/1) of hex in
aqueous solution (Naishstein and Lisovskaya, 1965) . Animals re-
ceiving the highest dosage, 2.0 ug/kg (40 ug/1), showed question-
able neutropenia and lymphocytosis which the investigators thought
possibly attributable to mobilization of the protective forces of
the organism in response to this dose.
Naishstein and Lisovskaya (1965) determined the lowest concen-
trations of hex capable of altering the smell and aftertaste of
water. Hex solutions were prepared by successive dilution of a
saturated aqueous solution of hex (20 mg/1). This stock solution
was prepared from dechlorinated tap water. The intensity of smell
and aftertaste was determined from 16 and 12 observations, respec-
tively; no indication of the number of experimental subjects was
given, however. The lower confidence limit of the mean threshold
response concentration was 1.4 ug/1 for smell and 1.6 yg/1 for
aftertaste. No other experimental details were presented. Based
C-64
-------
on these organoleptic effects, these investigators proposed a maxi-
mum permissible concentration of 1 ug/1. Stokinger and Woodward
(1975) themselves noted that oftentimes "other factors, including
taste, odor and color may outweigh health considerations because
acceptable limits for these may be well below the estimated health
limit."
Because chronic effects in a mammalian species (rats) have
been documented at water concentrations of hex as low as 40 ug/1,
it is obvious that an acceptable water quality criterion should be
well below this level. Thus, a reasonable safety factor of 10 to
100 applied to 40 ug/1 would place an appropriate criterion recom-
mendation in the range of 4.0-0.4 ug/1 in water. The level
recommended by Naishstein and Lisovskaya (1965) based on smell and
aftertaste falls well within this range.
No adverse effects on humans or mammals have been reported to
be caused by hex concentrations lower than approximately 1.0 ug/1.
Therefore, based on avoidance of alteration in smell and aftertaste
in water, a criterion of 1.0 ug/1 of hex in water is tentatively
suggested. It is to be stressed that this criterion is based on
inadequate chronic effects data and should be reevaluated upon com-
pletion of chronic oral toxicity studies.
C-65
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C-66
-------
Industrial Bio-Test Laboratories, Inc. 1977. Mutagenicity of PCL-
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C-67
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C-68
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